Sandwich assays in droplets

ABSTRACT

The invention generally relates to performing sandwich assays in droplets. In certain embodiments, the invention provides methods for detecting a target analyte that involve forming a compartmentalized portion of fluid including a portion of a sample suspected of containing a target analyte and a sample identifier, a first binding agent having a target identifier, and a second binding agent specific to the target analyte under conditions that produce a complex of the first and second binding agents with the target analyte, separating the complexes, and detecting the complexes, thereby detecting the target analyte.

RELATED APPLICATIONS

The present application claims the benefit of and priority to U.S.provisional patent application Ser. No. 61/388,413, filed Sep. 30, 2010,the content of which is incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The invention generally relates to performing sandwich assays,advantageously in droplets.

BACKGROUND

Biomarkers are commonly used to monitor and diagnosis disease.Biomarkers include nucleic acids, proteins, or other biologicalmolecules. Typically, an assay to identify a disease-associatedbiomarker is conducted in biological media, such as human tissues, cellsor fluids, and may be used to identify pathological processes beforeindividuals become symptomatic or to identify individuals who aresusceptible to diseases or already show signs and symptoms of a disease.

Standard screening assays have been developed that can detect bacteriaor viruses. Similarly, standard screening assays have been developedthat can use biomarkers to assess the health status of a patient and toprovide insight into the patient's risk of having a particular diseaseor disorder. An exemplary class of screening assays are sandwich assay.In a sandwich assay, a first binding agent with specificity for a targetanalyte (e.g., a bacteria, virus, or biomarker) is bound to a solidsupport. A sample is introduced to the solid support such that targetanalyte in the sample binds the first binding agent, thus becomingimmobilized to the solid support. Then, a second binding agent withspecificity for a target analyte is introduced to the and allowed tobind to the immobilized target analyte. The assay is named a sandwichassay because the first and second binding agents now sandwich thetarget analyte. A wash step is performed to remove unbound components ofthe sample and any excess binding agents. The second binding agenttypically includes a detectable label, and the label on the secondbinding agent is then detected, thus detecting the target analyte in thesample. Sandwich assays are typically antibody based and a commonly usedsandwich assay is an enzyme-linked immunosorbent assay (ELISA).

A problem with sandwich assays, particularly antibody based sandwichassays, is that they are unable to scale to high-level multiplexing.Issues of antibody cross-reactivity and non-specific adsorption occurwhen assays are multiplexed in the same tube. The ability to multiplexsamples, i.e., pool different patient samples, is important fordecreasing costs and increasing the through-put of analysis platforms.Additionally, assay development requires significant effort to optimizereagents to retain similar sensitivity as in single-plex assays.Further, such assays are not practical for use with small sample amountscollected at clinics.

SUMMARY

The invention utilizes microfluidics and droplet technology incombination with sandwich assays. Methods of the invention avoid theissues of antibody cross-reactivity and non-specific adsorption thatoccur when assays are multiplexed in bulk format. The use of dropletsallows high levels of multiplexing while retaining the specificity ofsingle-plex assays without the need for large sample volumes.

Methods of the invention involve forming a droplet that includesreagents for a sandwich assay (e.g., a first target binding agent havinga differentially detectable identifier and a second target bindingagent). Any technique known in the art for forming droplets may be usedwith methods of the invention. An exemplary method involves flowing astream of reagent fluid such that it intersects two opposing streams offlowing carrier fluid. The carrier fluid is immiscible with the reagentfluid. Intersection of the reagent fluid with the two opposing streamsof flowing carrier fluid results in partitioning of the reagent fluidinto individual reagent droplets. The carrier fluid may be any fluidthat is immiscible with the reagent fluid. An exemplary carrier fluid isoil. In certain embodiments, the carrier fluid includes a surfactant,such as a fluorosurfactant.

A sample containing target analyte (e.g., bacteria, virus, nucleic acidor protein) is introduced into a reagent droplet. This can occur byforming sample droplets and merging the sample droplets with the reagentdroplets to form mixed droplets that include sample and reagents for thesandwich assay. Another technique involves contacting the reagentdroplet with a fluid stream including the sample. Contact between thedroplet and the fluid stream results in a portion of the fluid streamintegrating with the droplet to form the mixed droplet.

Methods of the invention may be conducted in microfluidic channels. Assuch, in certain embodiments, methods of the invention may furtherinvolve flowing the droplet through a first channel and flowing thefluid stream through a second channel. The first and second channels areoriented such that the channels intersect each other. Any angle thatresults in an intersection of the channels may be used. In a particularembodiment, the first and second channels are oriented perpendicular toeach other. Methods of the invention may further involve applying anelectric field to the droplet and the fluid stream. The electric fieldassists in rupturing the interface separating the two sample fluids. Inparticular embodiments, the electric field is a high-frequency electricfield.

After forming the mixed droplet, a sandwich assay is conducted in thedroplet such that complexes of target analyte and first and secondbinding agents are formed. In certain embodiments, the assay isconducted in the presence of a competitive inhibitor. The competitiveinhibitor has affinity to analytes in the sample that may compete forbinding with the target analyte. The competitive inhibitor binds thesecompeting analytes and ensures that they do not compete with the targetanalyte for binding to the binding agents.

Generally, the second binding agent is configured such that it can becoupled to a solid support. For example, a terminal portion of thesecond binding agent may be functionalized with a terminal amine suchthat it can covalently bind an epoxide coated surface. Alternatively, aterminal end of the second binding agent is functionalized with onemember of a binding pair while a surface of the solid support is coatedwith the other member of the binding pair (e.g., biotin/avidin;biotin/streptavidin/ or digoxigenin/anti-digoxigenin). The support maybe a bead that is present in the droplet or it may be a substrateoutside of the droplet. Generally, the complexes become immobilized onthe solid support while uncomplexed sample components remain unbound inthe sample.

Bead-bound complexes can be released from the droplets and separatedfrom the unbound sample components. Alternatively, the droplet contentsare released and the complexes become immobilized to a solid support. Awash step is performed to remove the unbound sample components, and thenthe target identifier associated with the first binding agent isdetected.

The target identifier may be any type of differentially-detectableidentifier, such as an optically detectable label (e.g., fluorescent orchemiluminescent label), radiolabel, electrochemical label, or a barcodelabel. Detection may be by any methods known in the art and thedetection method will depend on the type of identifier used. Theidentifier may be releasably attached to the first binding agent or maybe irreversibly attached to the first binding agent.

In particular embodiments, the identifier is a barcode sequence. Thebarcode sequences can be released from the first binding agents and thenattached to each other to produce a single nucleic acid strand. Thisstrand is then amplified (e.g., rolling circle amplification or PCR) andthe amplified products are sequenced.

Sequencing may be by any method known in the art. In certainembodiments, sequencing is sequencing by synthesis. In otherembodiments, sequencing is single molecule sequencing by synthesis. Incertain embodiments, sequencing involves hybridizing a primer to thetemplate to form a template/primer duplex, contacting the duplex with apolymerase enzyme in the presence of a detectably labeled nucleotidesunder conditions that permit the polymerase to add nucleotides to theprimer in a template-dependent manner, detecting a signal from theincorporated labeled nucleotide, and sequentially repeating thecontacting and detecting steps at least once, wherein sequentialdetection of incorporated labeled nucleotide determines the sequence ofthe nucleic acid. Exemplary detectable labels include radiolabels,florescent labels, enzymatic labels, etc. In particular embodiments, thedetectable label may be an optically detectable label, such as afluorescent label. Exemplary fluorescent labels include cyanine,rhodamine, fluorescien, coumarin, BODIPY, alexa, or conjugatedmulti-dyes.

Another aspect of the invention provides reagent droplet libraries. Suchlibraries include a plurality of droplets containing the elementsnecessary for a sandwich assay prior to introduction of the targetanalyte. Preferably, droplets are surrounded by an immiscible carrierfluid, e.g., aqueous droplets surrounded by oil. Each droplet includes afirst binding agent having a differentially detectable identifier and asecond binding agent. The binding agents are any molecules that can binda target analyte in a sample. Exemplary binding agents include DNA, RNA,LNA, PNA, proteins, antibodies, or aptamers. Each droplet may furtherinclude a sample identifier that can bind to the identifier linked tothe first binding agent. In this manner, each droplet includes anidentifier for a particular target analyte and an identifier for aspecific droplet. Each droplet may further include a competitiveinhibitor.

Other aspects and advantages of the invention are apparent uponconsideration of the following detailed description thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing a device for droplet formation.

FIG. 2 is a drawing showing a device for droplet formation.

FIG. 3 depicts droplet generation, merging, and combining of droplets inan embodiment of the invention.

FIG. 4 depicts droplet library formation in an embodiment of theinvention.

FIG. 5 is a schematic illustrating an embodiment of the invention forpreparing, attaching, and releasing identifiers for analyzing targetanalytes.

FIG. 6 depicts target analyte sandwiches with identifiers embodyingprinciples of the invention.

FIG. 7 depicts a competitive inhibitor from one embodiment of theinvention.

FIG. 8 depicts introducing a sample identifier to a target analyteidentifier in one embodiment of the invention.

FIG. 9 depicts binding of a sample identifier to a target analyteidentifier in one embodiment of the invention.

DETAILED DESCRIPTION

The invention generally relates to sandwich assays in droplets. Incertain aspects, the invention provides methods for detecting andoptionally quantifying a target analyte by forming a compartmentalizedportion of fluid including a portion of a sample suspected of containinga target analyte, a first binding agent specific to the target analyteand having a target identifier, and a second binding agent specific to adifferent part of the target analyte under conditions that produce acomplex of the first and second binding agents with the target analyte,separating the complexes from uncomplexed target identifiers, anddetecting the complexes thereby detecting the target analyte. Theinvention allows for a high degree of multiplexing, thus allowing theuse of multiple samples, targets or both. Moreover, the invention isuseful to quantify targets as detailed below. There are numerousvariations in terms of the manner in which devices and methods of theinvention operate. A number of non-limiting examples are provided below.However, it is clear to one of skill in the art that numerous additionaladvantages and features of the invention are apparent upon considerationof the present specification and the examples that follow.

Samples

One of skill in the art will recognize that methods and systems of theinvention are not limited to any particular type of sample, and methodsand systems of the invention may be used with any type of organic,inorganic, or biological molecule. In particular embodiments the sampleincludes nucleic acid target molecules. Nucleic acid molecules can besynthetic or derived from naturally occurring sources. In oneembodiment, nucleic acid molecules are isolated from a biological samplecontaining a variety of other components, such as proteins, lipids andnon-template nucleic acids. Nucleic acid target molecules can beobtained from any cellular material, obtained from an animal, plant,bacterium, fungus, or any other cellular organism. In certainembodiments, the nucleic acid target molecules are obtained from asingle cell. Biological samples for use in the present invention includeviral particles or preparations. Nucleic acid target molecules can beobtained directly from an organism or from a biological sample obtainedfrom an organism, e.g., from blood, urine, cerebrospinal fluid, seminalfluid, saliva, sputum, stool and tissue. Any tissue or body fluidspecimen may be used as a source for nucleic acid for use in theinvention. Nucleic acid target molecules can also be isolated fromcultured cells, such as a primary cell culture or a cell line. The cellsor tissues from which target nucleic acids are obtained can be infectedwith a virus or other intracellular pathogen. A sample can also be totalRNA extracted from a biological specimen, a cDNA library, viral, orgenomic DNA.

Generally, nucleic acid can be extracted from a biological sample by avariety of techniques such as those described by Maniatis, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp.280-281 (1982). Nucleic acid molecules may be single-stranded,double-stranded, or double-stranded with single-stranded regions (forexample, stem- and loop-structures).

Nucleic acid obtained from biological samples typically is fragmented toproduce suitable fragments for analysis. Target nucleic acids may befragmented or sheared to desired length, using a variety of mechanical,chemical and/or enzymatic methods. DNA may be randomly sheared viasonication, e.g. Covaris method, brief exposure to a DNase, or using amixture of one or more restriction enzymes, or a transposase or nickingenzyme. RNA may be fragmented by brief exposure to an RNase, heat plusmagnesium, or by shearing. The RNA may be converted to cDNA. Iffragmentation is employed, the RNA may be converted to cDNA before orafter fragmentation. In one embodiment, nucleic acid from a biologicalsample is fragmented by sonication. In another embodiment, nucleic acidis fragmented by a hydroshear instrument. Generally, individual nucleicacid target molecules can be from about 40 bases to about 40 kb. Nucleicacid molecules may be single-stranded, double-stranded, ordouble-stranded with single-stranded regions (for example, stem- andloop-structures).

A biological sample as described herein may be homogenized orfractionated in the presence of a detergent or surfactant. Theconcentration of the detergent in the buffer may be about 0.05% to about10.0%. The concentration of the detergent can be up to an amount wherethe detergent remains soluble in the solution. In one embodiment, theconcentration of the detergent is between 0.1% to about 2%. Thedetergent, particularly a mild one that is nondenaturing, can act tosolubilize the sample. Detergents may be ionic or nonionic. Examples ofnonionic detergents include triton, such as the Triton® X series(Triton® X-100 t-Oct-C₆H₄—(OCH₂—CH₂)_(x)OH, x=9-10, Triton® X-100R,Triton® X-114 x=7-8), octyl glucoside, polyoxyethylene(9)dodecyl ether,digitonin, IGEPAL® CA630 octylphenyl polyethylene glycol,n-octyl-beta-D-glucopyranoside (betaOG), n-dodecyl-beta, Tween® 20polyethylene glycol sorbitan monolaurate, Tween® 80 polyethylene glycolsorbitan monooleate, polidocanol, n-dodecyl beta-D-maltoside (DDM),NP-40 nonylphenyl polyethylene glycol, C12E8 (octaethylene glycoln-dodecyl monoether), hexaethyleneglycol mono-n-tetradecyl ether(C14E06), octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG),Emulgen, and polyoxyethylene 10 lauryl ether (C12E10). Examples of ionicdetergents (anionic or cationic) include deoxycholate, sodium dodecylsulfate (SDS), N-lauroylsarcosine, and cetyltrimethylammoniumbromide(CTAB). A zwitterionic reagent may also be used in the purificationschemes of the present invention, such as Chaps, zwitterion 3-14, and3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulf-onate. It iscontemplated also that urea may be added with or without anotherdetergent or surfactant.

Lysis or homogenization solutions may further contain other agents, suchas reducing agents. Examples of such reducing agents includedithiothreitol (DTT), .beta.-mercaptoethanol, DTE, GSH, cysteine,cysteamine, tricarboxyethyl phosphine (TCEP), or salts of sulfurousacid.

Size selection of the nucleic acids may be performed to remove veryshort fragments or very long fragments. The nucleic acid fragments canbe partitioned into fractions comprising a desired number of fragmentsusing any suitable method known in the art. Suitable methods to limitthe fragment size in each fragment are known in the art. In variousembodiments of the invention, the fragment size is limited to betweenabout 10 and about 100 Kb or longer.

In another embodiment, the sample includes individual target proteins,protein complexes, proteins with translational modifications, andprotein/nucleic acid complexes. Protein targets include peptides, andalso include enzymes, hormones, structural components such as viralcapsid proteins, and antibodies. Protein targets may be synthetic orderived from naturally-occurring sources. In one embodiment of theinvention protein targets are isolated from biological samplescontaining a variety of other components including lipids, non-templatenucleic acids, and nucleic acids. In certain embodiments, proteintargets may be obtained from an animal, bacterium, fungus, cellularorganism, and single cells. Protein targets may be obtained directlyfrom an organism or from a biological sample obtained from the organism,including bodily fluids such as blood, urine, cerebrospinal fluid,seminal fluid, saliva, sputum, stool and tissue. Protein targets mayalso be obtained from cell and tissue lysates and biochemical fractions.An individual protein is an isolated polypeptide chain. A proteincomplex includes two or polypeptide chains. Samples may include proteinswith post translational modifications including but not limited tophosphorylation, methionine oxidation, deamidation, glycosylation,ubiquitination, carbamylation, s-carboxymethylation, acetylation, andmethylation. Protein/nucleic acid complexes include cross-linked orstable protein-nucleic acid complexes.

Extraction or isolation of individual proteins, protein complexes,proteins with translational modifications, and protein/nucleic acidcomplexes is performed using methods known in the art.

The invention is useful to detect and/or quantify other targetmolecules, such as any molecule that can be specifically bound in atleast two distinct portions of the target or any molecule in complexwith at least one other molecule that can be specifically bound bybinding agents.

Droplet Formation

Methods of the invention involve forming sample droplets. The dropletsare aqueous droplets that are surrounded by an immiscible carrier fluid.Methods of forming such droplets are shown for example in Link et al.(U.S. patent application numbers 2008/0014589, 2008/0003142, and2010/0137163), Stone et al. (U.S. Pat. No. 7,708,949 and U.S. patentapplication number 2010/0172803), Anderson et al. (U.S. Pat. No.7,041,481 and which reissued as RE41,780) and European publicationnumber EP2047910 to Raindance Technologies Inc. The content of each ofwhich is incorporated by reference herein in its entirety.

FIG. 1 shows an exemplary embodiment of a device 100 for dropletformation. Device 100 includes an inlet channel 101, and outlet channel102, and two carrier fluid channels 103 and 104. Channels 101, 102, 103,and 104 meet at a junction 105. Inlet channel 101 flows sample fluid tothe junction 105. Carrier fluid channels 103 and 104 flow a carrierfluid that is immiscible with the sample fluid to the junction 105.Inlet channel 101 narrows at its distal portion wherein it connects tojunction 105 (See FIG. 2). Inlet channel 101 is oriented to beperpendicular to carrier fluid channels 103 and 104. Droplets are formedas sample fluid flows from inlet channel 101 to junction 105, where thesample fluid interacts with flowing carrier fluid provided to thejunction 105 by carrier fluid channels 103 and 104. Outlet channel 102receives the droplets of sample fluid surrounded by carrier fluid.

Another approach to merging sample fluids involves forming a droplet,and contacting the droplet with a fluid stream, in which a portion ofthe fluid stream integrates with the droplet to form a mixed droplet. Inthis approach, only one phase needs to reach a merge area in a form of adroplet.

A reagent droplet, or library of reagent droplets is formed as describedabove, and can be stored in a collection of other droplets for combiningwith samples after re-introduction into a microfluidic device. Afterformation of the reagent droplet, the droplet is contacted with a flowof a sample fluid stream. Contact between the reagent droplet and thefluid stream results in a portion of the sample fluid stream integratingwith the reagent droplet to form a mixed droplet.

The monodisperse reagent droplets flow through a first channel separatedfrom each other by immiscible carrier fluid and suspended in theimmiscible carrier fluid. The droplets are delivered to the merge area,i.e., junction of the first channel with the second channel, by apressure-driven flow generated by a positive displacement pump. Whiledroplet arrives at the merge area, a bolus of a sample fluid isprotruding from an opening of the second channel into the first channel.Preferably, the channels are oriented perpendicular to each other.However, any angle that results in an intersection of the channels maybe used.

The bolus of the sample fluid stream continues to increase in size dueto pumping action of a positive displacement pump connected to channel,which outputs a steady stream of the second sample fluid into the mergearea. The flowing reagent droplet eventually contacts the bolus of thesample fluid that is protruding into the first channel. Contact betweenthe two fluids results in a portion of the sample fluid being segmentedfrom the sample fluid stream and joining with the reagent fluid dropletto form a mixed droplet. In certain embodiments, each incoming dropletof reagent fluid is merged with the same amount of sample fluid.

In certain embodiments, an electric charge is applied to the first andsecond sample fluids. Description of applying electric charge to samplefluids is provided in Link et al. (U.S. patent application number2007/0003442) and European Patent Number EP2004316 to RaindanceTechnologies Inc, the content of each of which is incorporated byreference herein in its entirety. Electric charge may be created usingany suitable technique, for example, by placing the reagent droplet andthe sample fluid within an electric field (which may be AC, DC, etc.),and/or causing a reaction to occur that causes the reagent droplet andthe sample fluid to have an electric charge, for example, a chemicalreaction, an ionic reaction, a photocatalyzed reaction, etc.

The electric field, in some embodiments, is generated from an electricfield generator, i.e., a device or system able to create an electricfield that can be applied to the fluid. The electric field generator mayproduce an AC field (i.e., one that varies periodically with respect totime, for example, sinusoidally, sawtooth, square, etc.), a DC field(i.e., one that is constant with respect to time), a pulsed field, etc.The electric field generator may be constructed and arranged to createan electric field within a fluid contained within a channel or amicrofluidic channel. The electric field generator may be integral to orseparate from the fluidic system containing the channel or microfluidicchannel, according to some embodiments.

Techniques for producing a suitable electric field (which may be AC, DC,etc.) are known to those of ordinary skill in the art. For example, inone embodiment, an electric field is produced by applying voltage acrossa pair of electrodes, which may be positioned on or embedded within thefluidic system (for example, within a substrate defining the channel ormicrofluidic channel), and/or positioned proximate the fluid such thatat least a portion of the electric field interacts with the fluid. Theelectrodes can be fashioned from any suitable electrode material ormaterials known to those of ordinary skill in the art, including, butnot limited to, silver, gold, copper, carbon, platinum, copper,tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as wellas combinations thereof. In some cases, transparent or substantiallytransparent electrodes can be used.

The electric field facilitates rupture of the interface separating thesample fluid and the droplet. Rupturing the interface facilitatesmerging of bolus of the s sample fluid and the reagent droplet. Theforming mixed droplet continues to increase in size until it a portionof the sample fluid breaks free or segments from the sample fluid streamprior to arrival and merging of the next reagent droplet. The segmentingof the portion of the sample fluid from the sample fluid stream occursas soon as the shear force exerted on the forming mixed droplet by theimmiscible carrier fluid overcomes the surface tension whose action isto keep the segmenting portion of the sample fluid connected with thesecond sample fluid stream. The now fully formed mixed droplet continuesto flow through the first channel.

The sample fluid is typically an aqueous buffer solution, such asultrapure water (e.g., 18 mega-ohm resistivity, obtained, for example bycolumn chromatography), 10 mM Tris HCl and 1 mM EDTA (TE) buffer,phosphate buffer saline (PBS) or acetate buffer. Any liquid or bufferthat is physiologically compatible with enzymes can be used. The carrierfluid is one that is immiscible with the sample fluid. The carrier fluidcan be a non-polar solvent, decane (e.g., tetradecane or hexadecane),fluorocarbon oil, silicone oil or another oil (for example, mineraloil).

In certain embodiments, the carrier fluid contains one or moreadditives, such as agents which reduce surface tensions (surfactants).Surfactants can include Tween, Span, fluorosurfactants, and other agentsthat are soluble in oil relative to water. In some applications,performance is improved by adding a second surfactant to the samplefluid. Surfactants can aid in controlling or optimizing droplet size,flow and uniformity, for example by reducing the shear force needed toextrude or inject droplets into an intersecting channel. This can affectdroplet volume and periodicity, or the rate or frequency at whichdroplets break off into an intersecting channel. Furthermore, thesurfactant can serve to stabilize aqueous emulsions in fluorinated oilsfrom coalescing.

In certain embodiments, the droplets may be coated with a surfactant.Preferred surfactants that may be added to the carrier fluid include,but are not limited to, surfactants such as sorbitan-based carboxylicacid esters (e.g., the “Span” surfactants, Fluka Chemika), includingsorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40),sorbitan monostearate (Span 60) and sorbitan monooleate (Span 80), andperfluorinated polyethers (e.g., DuPont Krytox 157 FSL, FSM, and/orFSH). Other non-limiting examples of non-ionic surfactants which may beused include polyoxyethylenated alkylphenols (for example, nonyl-,p-dodecyl-, and dinonylphenols), polyoxyethylenated straight chainalcohols, polyoxyethylenated polyoxypropylene glycols,polyoxyethylenated mercaptans, long chain carboxylic acid esters (forexample, glyceryl and polyglycerl esters of natural fatty acids,propylene glycol, sorbitol, polyoxyethylenated sorbitol esters,polyoxyethylene glycol esters, etc.) and alkanolamines (e.g.,diethanolamine-fatty acid condensates and isopropanolamine-fatty acidcondensates).

In certain embodiments, the carrier fluid may be caused to flow throughthe outlet channel so that the surfactant in the carrier fluid coats thechannel walls. In one embodiment, the fluorosurfactant can be preparedby reacting the perflourinated polyether DuPont Krytox 157 FSL, FSM, orFSH with aqueous ammonium hydroxide in a volatile fluorinated solvent.The solvent and residual water and ammonia can be removed with a rotaryevaporator. The surfactant can then be dissolved (e.g., 2.5 wt %) in afluorinated oil (e.g., Flourinert (3M)), which then serves as thecarrier fluid.

The oil can comprise at least one fluorosurfactant. In some embodiments,the fluorosurfactant comprised within immiscible fluorocarbon oil is ablock copolymer consisting of one or more perfluorinated polyether(PFPE) blocks and one or more polyethylene glycol (PEG) blocks. In otherembodiments, the fluorosurfactant is a triblock copolymer consisting ofa PEG center block covalently bound to two PFPE blocks by amide linkinggroups. The presence of the fluorosurfactant (similar to uniform size ofthe droplets in the library) may be important to maintain the stabilityand integrity of the droplets and may also be beneficial for thesubsequent use of the droplets within the library for the variousbiological and chemical assays described herein. Fluids (e.g., aqueousfluids, immiscible oils, etc.) and other surfactants that can beutilized in the droplet libraries of the present invention are describedin greater detail herein.

Microfluidic Systems

Reagents can be reformatted as droplet libraries utilizing automateddevices. Specifically, the library element components can be placed ontoplates containing any number of wells, i.e. 96, 384, etc. The plates canthen be placed in any one of a number of devices known in the art forforming the droplets. The droplets can be placed into a vial or othersuch container, containing the stable droplet library for later use. Ingeneral, the process aspirates the components from each well of a wellplate and infuses them through tubing connected to a microfluidic device(described in greater detail herein) which can be used to form thedroplets that constitute a single library member or ‘element’. Thetubing is rinsed at a wash station and then the process can be repeatedto generate droplets for the next library element.

A collection vial can be used to contain the droplets made using theAutomated Droplet Library Production. In one example, the collectionvial has two holes, a first hole in the center of the vial cap and asecond hole part way to the edge of the vial cap. The vial is firstfilled with oil through the second hole to purge air out first hole, theemulsion is then introduced to the vial through the first hole,typically this is done sequentially one library element at a time at lowvolume fraction, and oil is displaced and goes out through the secondhole. The collected droplets can be stored in the vial for periods oftime in excess of 3 months. To remove the emulsion for use or to makesmaller aliquots, oil is introduced through the second opening todisplace the emulsion and drive out the first opening.

The droplet libraries of the present invention are preferably formed byutilizing microfluidic devices and are preferably utilized to performvarious biological and chemical assays on microfluidic devices, asdescribed in detail herein. The present invention also provides embeddedmicrofluidic nozzles. In order to create a monodisperse (<1.5%polydispersity) emulsion directly from a library well, a nozzle can beformed directly into the fitting used to connect the storagewell/reservoir (e.g. syringe) to a syringe tip (e.g. capillary tubing).Examples of suitable nozzles to create the droplet libraries of theinstant invention are described in WO 2007/081385 and WO 2008/063227.

Since the flow is three dimensional, under this design surface wettingeffects are minimized. The nozzle can be made from one or two oil linesproviding constant flow of oil into the nozzle, a connection to thecapillary tubing, and a connection to the storage well/reservoir (e.g.syringe). The high resolution part of the nozzle can be made out of asmall bore tubing or a small, simple part molded or stamped from anappropriate material (Teflon®, plastic, metal, etc). If necessary, thenozzle itself could be formed into the tip of the ferrule using postmold processing such as laser ablation or drilling.

This nozzle design eliminates the surface wetting issues surrounding thequasi-2D flow associated with typical microfluidic nozzles made usingsoft lithography or other standard microfluidic chip manufacturingtechniques. This is because the nozzle design is fully 3-dimensional,resulting is a complete isolation of the nozzle section from thecontinuous aqueous phase. This same design can also be used forgeneration of emulsions required for immediate use, where the aqueousline would be attached directly to a syringe and the outlet of thenozzle would be used to transport the emulsion to the point of use (e.g.into a microfluidic PCR chip, delay line, etc).

In another embodiment, the present invention provides compositions andmethods to directly emulsify library elements from standard librarystorage geometries (e.g. 96 well plates, etc). In order to create amonodisperse emulsion from fluids contained in a library well plate,this invention would include microfluidic based nozzles manufacturedsimultaneously with an appropriately designed fluidic interconnect orwell.

Specifically, the microfluidic devices and methods described herein arebased on the creation and electrical manipulation of aqueous phasedroplets (e.g., droplet libraries) completely encapsulated by an inertimmiscible oil stream. This combination enables precise dropletgeneration, highly efficient, electrically addressable, dropletcoalescence, and controllable, electrically addressable single dropletsorting. The microfluidic devices include one or more channels andmodules. The integration of these modules is an essential enablingtechnology for a droplet based, high-throughput microfluidic reactorsystem and provides an ideal system for utilizing the droplet librariesprovided herein for numerous biological, chemical, or diagnosticapplications.

Substrates

The microfluidic device of the present invention includes one or moreanalysis units. An “analysis unit” is a micro substrate, e.g., amicrochip. The terms microsubstrate, substrate, microchip, and chip areused interchangeably herein. The analysis unit includes at least oneinlet channel, at least one main channel and at least one inlet module.The analysis unit can further include at least one coalescence module.at least one detection module and one or more sorting modules. Thesorting module can be in fluid communication with branch channels whichare in fluid communication with one or more outlet modules (collectionmodule or waste module). For sorting applications, at least onedetection module cooperates with at least one sorting module to divertflow via a detector-originated signal. It shall be appreciated that the“modules” and “channels” are in fluid communication with each other andtherefore may overlap; i.e., there may be no clear boundary where amodule or channel begins or ends. A plurality of analysis units of theinvention may be combined in one device. The dimensions of the substrateare those of typical microchips, ranging between about 0.5 cm to about15 cm per side and about 1 micron to about 1 cm in thickness. Theanalysis unit and specific modules are described in further detail in WO2006/040551; WO 2006/040554; WO 2004/002627; WO 2004/091763; WO2005/021151; WO 2006/096571; WO 2007/089541; WO 2007/081385 and WO2008/063227.

A variety of materials and methods can be used to form any of thedescribed components of the systems and devices of the invention. Forexample, various components of the invention can be formed from solidmaterials, in which the channels can be formed via molding,micromachining, film deposition processes such as spin coating andchemical vapor deposition, laser fabrication, photolithographictechniques, etching methods including wet chemical or plasma processes,and the like. See, for example, Angell, et al., Scientific American,248:44-55, 1983. At least a portion of the fluidic system can be formedof silicone by molding a silicone chip. Technologies for precise andefficient formation of various fluidic systems and devices of theinvention from silicone are known. Various components of the systems anddevices of the invention can also be formed of a polymer, for example,an elastomeric polymer such as polydimethylsiloxane (“PDMS”),polytetrafluoroethylene (“PTFE”) or Teflon®, or the like, orthermoplastic polymers.

Silicone polymers are preferred, for example, the silicone elastomerpolydimethylsiloxane. Non-limiting examples of PDMS polymers includethose sold under the trademark Sylgard by Dow Chemical Co., Midland,Mich., and particularly Sylgard 182, Sylgard 184, and Sylgard 186.Silicone polymers including PDMS have several beneficial propertiessimplifying formation of the microfluidic structures of the invention.For instance, such materials are inexpensive, readily available, and canbe solidified from a prepolymeric liquid via curing with heat. Forexample, PDMSs are typically curable by exposure of the prepolymericliquid to temperatures of about, for example, about 65° C. to about 75°C. for exposure times of, for example, about an hour. Also, siliconepolymers, such as PDMS, can be elastomeric and thus may be useful forforming very small features with relatively high aspect ratios,necessary in certain embodiments of the invention. Flexible (e.g.,elastomeric) molds or masters can be advantageous in this regard.

One advantage of forming structures such as microfluidic structures ofthe invention from silicone polymers, such as PDMS, is the ability ofsuch polymers to be oxidized, for example by exposure to anoxygen-containing plasma such as an air plasma, so that the oxidizedstructures contain, at their surface, chemical groups capable ofcross-linking to other oxidized silicone polymer surfaces or to theoxidized surfaces of a variety of other polymeric and non-polymericmaterials. Thus, components can be formed and then oxidized andessentially irreversibly sealed to other silicone polymer surfaces, orto the surfaces of other substrates reactive with the oxidized siliconepolymer surfaces, without the need for separate adhesives or othersealing means. In most cases, sealing can be completed simply bycontacting an oxidized silicone surface to another surface without theneed to apply auxiliary pressure to form the seal. That is, thepre-oxidized silicone surface acts as a contact adhesive againstsuitable mating surfaces. Specifically, in addition to beingirreversibly sealable to itself, oxidized silicone such as oxidized PDMScan also be sealed irreversibly to a range of oxidized materials otherthan itself including, for example, glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, andepoxy polymers, which have been oxidized in a similar fashion to thePDMS surface (for example, via exposure to an oxygen-containing plasma).Oxidation and sealing methods useful in the context of the presentinvention, as well as overall molding techniques, are described in theart, for example, in Duffy et al., “Rapid Prototyping of MicrofluidicSystems and Polydimethylsiloxane,” Anal. Chem., 70:474-480, 1998.

Another advantage to forming microfluidic structures of the invention(or interior, fluid-contacting surfaces) from oxidized silicone polymersis that these surfaces can be much more hydrophilic than the surfaces oftypical elastomeric polymers (where a hydrophilic interior surface isdesired). Such hydrophilic channel surfaces can thus be more easilyfilled and wetted with aqueous solutions than can structures comprisedof typical, unoxidized elastomeric polymers or other hydrophobicmaterials.

Channels

The microfluidic substrates of the present invention include channelsthat form the boundary for a fluid. A “channel,” as used herein, means afeature on or in a substrate that at least partially directs the flow ofa fluid. In some cases, the channel may be formed, at least in part, bya single component, e.g., an etched substrate or molded unit. Thechannel can have any cross-sectional shape, for example, circular, oval,triangular, irregular, square or rectangular (having any aspect ratio),or the like, and can be covered or uncovered (i.e., open to the externalenvironment surrounding the channel).

In embodiments where the channel is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, and/or the entire channel may be completely enclosed along itsentire length with the exception of its inlet and outlet. The channelsof the invention can be formed, for example by etching a silicon chipusing conventional photolithography techniques, or using amicromachining technology called “soft lithography” as described byWhitesides and Xia, Angewandte Chemie International Edition 37, 550(1998).

An open channel generally will include characteristics that facilitatecontrol over fluid transport, e.g., structural characteristics (anelongated indentation) and/or physical or chemical characteristics(hydrophobicity vs. hydrophilicity) and/or other characteristics thatcan exert a force (e.g., a containing force) on a fluid. The fluidwithin the channel may partially or completely fill the channel. In somecases the fluid may be held or confined within the channel or a portionof the channel in some fashion, for example, using surface tension(e.g., such that the fluid is held within the channel within a meniscus,such as a concave or convex meniscus). In an article or substrate, some(or all) of the channels may be of a particular size or less, forexample, having a largest dimension perpendicular to fluid flow of lessthan about 5 mm, less than about 2 mm, less than about 1 mm, less thanabout 500 microns, less than about 200 microns, less than about 100microns, less than about 60 microns, less than about 50 microns, lessthan about 40 microns, less than about 30 microns, less than about 25microns, less than about 10 microns, less than about 3 microns, lessthan about 1 micron, less than about 300 nm, less than about 100 nm,less than about 30 nm, or less than about 10 nm or less in some cases.

A “main channel” is a channel of the device of the invention thatpermits the flow of molecules, cells, small molecules or particles pasta coalescence module for coalescing one or more droplets, and, ifpresent, a detection module for detection (identification) ormeasurement of a droplet and a sorting module for sorting a dropletbased on the detection in the detection module. The main channel istypically in fluid communication with the coalescence, detection and/orsorting modules, as well as, an inlet channel of the inlet module. Themain channel is also typically in fluid communication with an outletmodule and optionally with branch channels, each of which may have acollection module or waste module. These channels permit the flow ofmolecules, cells, small molecules or particles out of the main channel.An “inlet channel” permits the flow of molecules, cells, small moleculesor particles into the main channel. One or more inlet channelscommunicate with one or more means for introducing a sample into thedevice of the present invention. The inlet channel communicates with themain channel at an inlet module.

The microfluidic substrate can also comprise one or more fluid channelsto inject or remove fluid in between droplets in a droplet stream forthe purpose of changing the spacing between droplets. The channels ofthe device of the present invention can be of any geometry as described.However, the channels of the device can comprise a specific geometrysuch that the contents of the channel are manipulated, e.g., sorted,mixed, prevent clogging, etc.

A microfluidic substrate can also include a specific geometry designedin such a manner as to prevent the aggregation of biological/chemicalmaterial and keep the biological/chemical material separated from eachother prior to encapsulation in droplets. The geometry of channeldimension can be changed to disturb the aggregates and break them apartby various methods, that can include, but is not limited to, geometricpinching (to force cells through a (or a series of) narrow region(s),whose dimension is smaller or comparable to the dimension of a singlecell) or a barricade (place a series of barricades on the way of themoving cells to disturb the movement and break up the aggregates ofcells). To prevent material (e.g., cells and other particles ormolecules) from adhering to the sides of the channels, the channels (andcoverslip, if used) may have a coating which minimizes adhesion. Thesurface of the channels of the microfluidic device can be coated withany anti-wetting or blocking agent for the dispersed phase. The channelcan be coated with any protein to prevent adhesion of thebiological/chemical sample. Channels can be coated by any means known inthe art. For example, the channels are coated with Teflon®, BSA,PEG-silane and/or fluorosilane in an amount sufficient to preventattachment and prevent clogging. In another example, the channels can becoated with a cyclized transparent optical polymer obtained bycopolymerization of perfluoro (alkenyl vinyl ethers), such as the typesold by Asahi Glass Co. under the trademark Cytop. In such an example,the coating is applied from a 0.1-0.5 wt % solution of Cytop CTL-809M inCT-Solv 180. This solution can be injected into the channels of amicrofluidic device via a plastic syringe. The device can then be heatedto about 90° C. for 2 hours, followed by heating at 200° C. for anadditional 2 hours. In another embodiment, the channels can be coatedwith a hydrophobic coating of the type sold by PPG Industries, Inc.under the trademark Aquapel (e.g., perfluoroalkylalkylsilane surfacetreatment of plastic and coated plastic substrate surfaces inconjunction with the use of a silica primer layer) and disclosed in U.S.Pat. No. 5,523,162. By fluorinating the surfaces of the channels, thecontinuous phase preferentially wets the channels and allows for thestable generation and movement of droplets through the device. The lowsurface tension of the channel walls thereby minimizes the accumulationof channel clogging particulates.

The surface of the channels in the microfluidic device can be alsofluorinated by any means known in the art to prevent undesired wettingbehaviors. For example, a microfluidic device can be placed in apolycarbonate dessicator with an open bottle of(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. The dessicatoris evacuated for 5 minutes, and then sealed for 20-40 minutes. Thedessicator is then backfilled with air and removed. This approach uses asimple diffusion mechanism to enable facile infiltration of channels ofthe microfluidic device with the fluorosilane and can be readily scaledup for simultaneous device fluorination.

Fluids

The fluids described herein are related to the fluids within the dropletlibraries and to the fluids within a microfluidic device. Themicrofluidic device of the present invention is capable of controllingthe direction and flow of fluids and entities within the device. Theterm “flow” means any movement of liquid or solid through a device or ina method of the invention, and encompasses without limitation any fluidstream, and any material moving with, within or against the stream,whether or not the material is carried by the stream. For example, themovement of molecules, beads, cells or virions through a device or in amethod of the invention, e.g. through channels of a microfluidic chip ofthe invention, comprises a flow. This is so, according to the invention,whether or not the molecules, beads, cells or virions are carried by astream of fluid also comprising a flow, or whether the molecules, cellsor virions are caused to move by some other direct or indirect force ormotivation, and whether or not the nature of any motivating force isknown or understood. The application of any force may be used to providea flow, including without limitation, pressure, capillary action,electro-osmosis, electrophoresis, dielectrophoresis, optical tweezers,and combinations thereof, without regard for any particular theory ormechanism of action, so long as molecules, cells or virions are directedfor detection, measurement or sorting according to the invention.Specific flow forces are described in further detail herein.

The flow stream in the main channel is typically, but not necessarily,continuous and may be stopped and started, reversed or changed in speed.A liquid that does not contain sample molecules, cells or particles canbe introduced into a sample inlet well or channel and directed throughthe inlet module, e.g., by capillary action, to hydrate and prepare thedevice for use. Likewise, buffer or oil can also be introduced into amain inlet region that communicates directly with the main channel topurge the device (e.g., or “dead” air) and prepare it for use. Ifdesired, the pressure can be adjusted or equalized, for example, byadding buffer or oil to an outlet module.

According to the invention, a fluidic stream may be continuous and/ordiscontinuous. A “continuous” fluidic stream is a fluidic stream that isproduced as a single entity, e.g., if a continuous fluidic stream isproduced from a channel, the fluidic stream, after production, appearsto be contiguous with the channel outlet. The continuous fluidic streamis also referred to as a continuous phase fluid or carrier fluid. Thecontinuous fluidic stream may be laminar (potentially including streamsof two or more fluids), or turbulent in some cases.

Similarly, a “discontinuous” fluidic stream is a fluidic stream that isnot produced as a single entity. The discontinuous fluidic stream isalso referred to as the dispersed phase fluid or sample fluid. Adiscontinuous fluidic stream may have the appearance of individualdroplets, optionally surrounded by a second fluid. The dispersed phasefluid can include a biological/chemical material. Thebiological/chemical material can be tissues, cells, particles, proteins,antibodies, amino acids, nucleotides, small molecules, andpharmaceuticals. The biological/chemical material can include one ormore labels known in the art. The label can be an optical label, anenzymatic label or a radioactive label. The label can be any detectablelabel, e.g., a protein, a DNA tag, a dye, a quantum dot or a radiofrequency identification tag, or combinations thereof. In someembodiments, the label is an optical label. The label can be detected byany means known in the art. Preferably, the label is detected byfluorescence polarization, fluorescence intensity, fluorescencelifetime, fluorescence energy transfer, pH, ionic content, temperatureor combinations thereof. Various labels and means for detection aredescribed in greater detail herein.

The term “emulsion” refers to a preparation of one liquid distributed insmall globules (also referred to herein as drops, droplets orNanoReactors) in the body of a second liquid. The first and secondfluids are immiscible with each other. For example, the discontinuousphase can be an aqueous solution and the continuous phase can ahydrophobic fluid such as an oil. This is termed a water in oilemulsion. Alternatively, the emulsion may be a oil in water emulsion. Inthat example, the first liquid, which is dispersed in globules, isreferred to as the discontinuous phase, whereas the second liquid isreferred to as the continuous phase or the dispersion medium. Thecontinuous phase can be an aqueous solution and the discontinuous phaseis a hydrophobic fluid, such as an oil (e.g., decane, tetradecane, orhexadecane). The droplets or globules of oil in an oil in water emulsionare also referred to herein as “micelles”, whereas globules of water ina water in oil emulsion may be referred to as “reverse micelles”.

The fluidic droplets may each be substantially the same shape and/orsize. The droplets may be uniform in size. The shape and/or size can bedetermined, for example, by measuring the average diameter or othercharacteristic dimension of the droplets. The “average diameter” of aplurality or series of droplets is the arithmetic average of the averagediameters of each of the droplets. Those of ordinary skill in the artwill be able to determine the average diameter (or other characteristicdimension) of a plurality or series of droplets, for example, usinglaser light scattering, microscopic examination, or other knowntechniques. The diameter of a droplet, in a non-spherical droplet, isthe mathematically-defined average diameter of the droplet, integratedacross the entire surface. The average diameter of a droplet (and/or ofa plurality or series of droplets) may be, for example, less than about1 mm, less than about 500 micrometers, less than about 200 micrometers,less than about 100 micrometers, less than about 75 micrometers, lessthan about 50 micrometers, less than about 25 micrometers, less thanabout 10 micrometers, or less than about 5 micrometers in some cases.The 15 average diameter may also be at least about 1 micrometer, atleast about 2 micrometers, at least about 3 micrometers, at least about5 micrometers, at least about 10 micrometers, at least about 15micrometers, or at least about 20 micrometers in certain cases.

As used herein, the term “NanoReactor” and its plural encompass theterms “droplet”, “nanodrop”, “nanodroplet”, “microdrop” or“microdroplet” as defined herein, as well as an integrated system forthe manipulation and probing of droplets, as described in detail herein.Nanoreactors as described herein can be 0.1-1000 μm (e.g., 0.1, 0.2 . .. 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 . . . 1000), or any size withinthis range. Droplets at these dimensions tend to conform to the size andshape of the channels, while maintaining their respective volumes. Thus,as droplets move from a wider channel to a narrower channel they becomelonger and thinner, and vice versa.

The microfluidic substrate of this invention most preferably generateround, highly uniform, monodisperse droplets (<1.5% polydispersity).Droplets and methods of forming monodisperse droplets in microfluidicchannels is described in WO 2006/040551; WO 2006/040554; WO 2004/002627;WO 2004/091763; WO 2005/021151; WO 2006/096571; WO 2007/089541; WO2007/081385 and WO 2008/063227. The droplet forming liquid is typicallyan aqueous buffer solution, such as ultrapure water (e.g., 18 mega-ohmresistivity, obtained, for example by column chromatography), 10 mM TrisHCl and 1 mM EDTA (TE) buffer, phosphate buffer saline (PBS) or acetatebuffer. Any liquid or buffer that is physiologically compatible with thepopulation of molecules, cells or particles to be analyzed and/or sortedcan be used. The fluid passing through the main channel and in which thedroplets are formed is one that is immiscible with the droplet formingfluid. The fluid passing through the main channel can be a non-polarsolvent, decane (e.g., tetradecane or hexadecane), fluorocarbon oil,silicone oil or another oil (for example, mineral oil).

The droplet may also contain biological/chemical material (e.g.,molecules, cells, or other particles) for combination, analysis and/orsorting in the device. The droplets of the dispersed phase fluid cancontain more than one particle or can contain no more than one particle.Droplets of a sample fluid can be formed within the inlet module on themicrofluidic device or droplets (or droplet libraries) can be formedbefore the sample fluid is introduced to the microfluidic device (stabledroplet libraries can be stored after manufacturing, for introductiononto the microfluidic device and combination with sample droplets orother droplet libraries). To permit effective interdigitation,coalescence and detection, the droplets comprising each sample to beanalyzed must be monodisperse. As described in more detail herein, inmany applications, different samples to be analyzed are contained withindroplets of different sizes. Droplet size must be highly controlled toensure that droplets containing the correct contents for analysis andcoalesced properly. As such, the present invention provides devices andmethods for forming droplets and droplet libraries.

Surfactants

The fluids used in the invention may contain one or more additives, suchas agents which reduce surface tensions (surfactants). Surfactants caninclude Tween, Span, fluorosurfactants, and other agents that aresoluble in oil relative to water. In some applications, performance isimproved by adding a second surfactant to the aqueous phase. Surfactantscan aid in controlling or optimizing droplet size, flow and uniformity,for example by reducing the shear force needed to extrude or injectdroplets into an intersecting channel. This can affect droplet volumeand periodicity, or the rate or frequency at which droplets break offinto an intersecting channel. Furthermore, the surfactant can serve tostabilize aqueous emulsions in fluorinated oils from coalescing. Thepresent invention provides compositions and methods to stabilize aqueousdroplets in a fluorinated oil and minimize the transport of positivelycharged reagents (particularly, fluorescent dyes) from the aqueous phaseto the oil phase. The droplets may be coated with a surfactant.Preferred surfactants that may be added to the continuous phase fluidinclude, but ate not limited to, surfactants such as sorbitan-basedcarboxylic acid esters (e.g., the “Span” surfactants, Fluka Chemika),including sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span40), sorbitan monostearate (Span 60) and sorbitan monooleate (Span 80),and perfluorinated polyethers (e.g., DuPont Krytox 157 FSL, FSM, and/orFSH). Other non-limiting examples of non-ionic surfactants which may beused include polyoxyethylenated alkylphenols (for example, nonyl-,p-dodecyl-, and dinonylphenols), polyoxyethylenated straight chainalcohols, polyoxyethylenated polyoxypropylene glycols,polyoxyethylenated mercaptans, long chain carboxylic acid esters (forexample, glyceryl and polyglycerl esters of natural fatty acids,propylene glycol, sorbitol, polyoxyethylenated sorbitol esters,polyoxyethylene glycol esters, etc.) and alkanolamines (e.g.,diethanolamine-fatty acid condensates and isopropanolamine-fatty acidcondensates). In addition, ionic surfactants such as sodium dodecylsulfate (SDS) may also be used. However, such surfactants are generallyless preferably for many embodiments of the invention. For instance, inthose embodiments where aqueous droplets are used as nanoreactors forchemical reactions (including biochemical reactions) or are used toanalyze and/or sort biomaterials, a water soluble surfactant such as SDSmay denature or inactivate the contents of the droplet.

The carrier fluid can be an oil (e.g., decane, tetradecane orhexadecane) or fluorocarbon oil that contains a surfactant (e.g., anon-ionic surfactant such as a Span surfactant) as an additive(preferably between about 0.2 and 5% by volume, more preferably about2%). A user can preferably cause the carrier fluid to flow throughchannels of the microfluidic device so that the surfactant in thecarrier fluid coats the channel walls.

Fluorocarbon oil continuous phases are well-suited as the continuousphase for aqueous droplet libraries for a number of reasons. Fluorousoils are both hydrophobic and lipophobic. Therefore, they have lowsolubility for components of the aqueous phase and they limit moleculardiffusion between droplets. Also, fluorous oils present an inertinterface for chemistry and biology within droplets. In contrast tohydrocarbon or silicone oils, fluorous oils do not swell PDMS materials,which is a convenient material for constructing microfluidic channels.Finally, fluorocarbon oils have good solubility for gases, which isnecessary for the viability of encapsulated cells.

Combinations of surfactant(s) and oils must be developed to facilitategeneration, storage, and manipulation of droplets to maintain the uniquechemical/biochemical/biological environment within each droplet of adiverse library. Therefore, the surfactant and oil combination must (1)stabilize droplets against uncontrolled coalescence during the dropforming process and subsequent collection and storage, (2) minimizetransport of any droplet contents to the oil phase and/or betweendroplets, and (3) maintain chemical and biological inertness withcontents of each droplet (e.g., no adsorption or reaction ofencapsulated contents at the oil-water interface, and no adverse effectson biological or chemical constituents in the droplets). In addition tothe requirements on the droplet library function and stability, thesurfactant-in-oil solution must be coupled with the fluid physics andmaterials associated with the platform. Specifically, the oil solutionmust not swell, dissolve, or degrade the materials used to construct themicrofluidic chip, and the physical properties of the oil (e.g.,viscosity, boiling point, etc.) must be suited for the flow andoperating conditions of the platform.

Droplets formed in oil without surfactant are not stable to permitcoalescence, so surfactants must be dissolved in the fluorous oil thatis used as the continuous phase for the emulsion library. Surfactantmolecules are amphiphilic—part of the molecule is oil soluble, and partof the molecule is water soluble. When a water-oil interface is formedat the nozzle of a microfluidic chip for example in the inlet moduledescribed herein, surfactant molecules that are dissolved in the oilphase adsorb to the interface. The hydrophilic portion of the moleculeresides inside the droplet and the fluorophilic portion of the moleculedecorates the exterior of the droplet. The surface tension of a dropletis reduced when the interface is populated with surfactant, so thestability of an emulsion is improved. In addition to stabilizing thedroplets against coalescence, the surfactant should be inert to thecontents of each droplet and the surfactant should not promote transportof encapsulated components to the oil or other droplets.

A very large body of fundamental research and commercial applicationdevelopment exists for non-fluorous surfactants and emulsions rangingfrom sub-micron droplets to very large, macroscopic emulsions. Incontrast, fundamental knowledge and commercial practice with fluorinatedoils and surfactants is much less common. More specifically, testing anddevelopment of fluorosurfactants and fluorous oil formulations for theapplication of creating large libraries of micron-scale droplets withunique composition is limited to only a few groups throughout the world.Only a few commercially-available fluorosurfactants that stabilizewater-in-fluorocarbon oil emulsions exist. For instance, surfactantswith short fluorotelomer-tails (typically perfluorinated C6 to C10) areavailable, but they do not provide sufficient long-term emulsionstability. Fluorosurfactants with longer fluorocarbon tails, such asperfluoropolyether (PFPE), are limited to molecules with ionicheadgroups.

Classes of oils are available from wide variety of fluorinated oils andare available from commercial sources. The requirements for the oil are(1) immiscibility with the aqueous phase, (2) solubility of emulsionstabilizing surfactants in the oil, and (3) compatibility and/orinsolubility of reagents from the droplet phase. Oils includehydrofluoroethers, which are fluorinated alkyl chains coupled withhydrocarbon chemistry through and ether bond. One supplier of this typeof oil is 3M.

The products are marketed as Novec Engineered Fluids or HFE-series oils.This oils include but are not limited to, HFE-7500, which is a preferredembodiment as it provides superior droplet stability seems to be higher.Other oils include FIFE-7100, -7200, -7600, which are examples of otherHFEs available from 3M. These can be used as stand-alone oils orcomponents of oil mixtures to optimize emulsion properties andperformance. Other hydrofluoroethers are also available from otherproducers, distributors, or resellers may offer hydrofluoroethers.Another class of oil is perfluoroalkylamines, which are perfluorinatedoils based on perfluoroalkyl amine structures. 3M produces these oils asFluorinert Electronic Liquids (FC-oils). Fluorinert products differ byvariations in alkyl chain length, branch structure, and combinations ofdifferent structures or pure oils. Many of them offer the potential forstand-alone oils or components of oil mixtures to optimize emulsionproperties and performance. Specific examples are Fluorinert FC3283,Fluorinert FC-40, which are a preferred embodiments. Higher viscosityand boiling point useful for applications requiring elevated temperature(e.g., thermocyling for PCR). Other Fluorinert series can be used forstand-alone oils or components of oil mixtures to optimize emulsionproperties and performance. Again, other perfluoroalkylamines areavailable from other producers, distributors, or resellers may offerperfluoroalkylamines.

Fluorinated organics/solvents offer a number of perfluorinated orpartially fluorinated solvents are available from a variety ofproducers, distributors, and/or resellers. Many of them offer thepotential for stand-alone oils or components of oil mixtures to optimizeemulsion properties and performance. Examples of fluorinated organicreagents utilized, included (but not limited to) trifluoroacetic acidand hexafluoroisopropanol, to improve droplet stability in otherfluorinated oil systems. Additionally, fluoropolymers may also be usedwithin a microfluidic system. Examples of fluoropolymers include, KrytoxGPL oils, Solvay Galden oils, and other liquid fluoropolymers. Otherfluorinated materials find widespread use in a variety of industries,but they are generally not wellknown in the disciplines of interfacial,colloidal, physical, or synthetic organic chemistry. Therefore, a numberof other candidates for oils exist in specialty and niche marketapplications. As such, new oils have been targeted partially that areper-fluorinated materials, which are not widely recognized.

The properties of oils selected are based upon their chemicalproperties, such as, among others molecular structure, fluorine contentand solvating strength. Physical properties of oils examined includeviscosity, boiling point, thermal expansion coefficient, oil-in-watersolubility, water-in-oil solubility, dielectric constant, polarity, andoil-in-water surface tension.

Classes of surfactants include fluorosurfactants that can be categorizedby the type of fluorophilic portion of the molecule, the type ofhydrophilic, or polar, portion, and the chemistry used to link thedifferent parts of the molecule. Materials developed are capable ofstabilizing an emulsion or droplet library. The preferred embodiment isthe EA surfactant. Specifically, the EA surfactant is aKrytox-PEG-Krytox. The EA surfactant is a nonionic tri-block copolymersurfactant was developed to avoid issues that the ionic surfactants(e.g., RR, see below) which result from the use of some other ionicsurfactant. Specifically, ionic surfactants interact with chargedspecies in the droplets and can sequester ions (e.g., magnesium requiredfor the PCR reaction) or other reagents to the oil phase. The structureof the EA surfactant comprises a PEG—approximately 600 Da with amine endfunctionality, PFPE—Mn is—5000-8000 from a Krytox FSH starting materialand the linker is an amide coupling. Another surfactant includes the RRsurfactant, which is a Krytox ammonium carboxylate. Alternativematerials are alternative fluorophilic portion, i.e., PFPE (Solvay orDemnum), Poly(fluoroalkylacrylate) and other non-polymeric and partiallyfluorinated materials. Alternative head-group chemistry for thehydrophilic portion includes, non-ionic head groups like PEG (Mw, Mw/Mn(PDI)) and functionality (i.e., diblock, triblock and dendritic). Othersinclude morpholino. Ionic head groups for the hydrophilic portioninclude anionic, such as elemental and amine salts and further cationichead portions. Other head group chemistries include zwitterionic, hybrid(e.g., PEG-ionic and zonyl FSO/FSN), lipophilic (e.g, lipophilic topromote bilayer and lipophilic spacer to hydrophile). Anotheralternative is alternative coupling chemistry such as,phosphoryl/Friedel-Crafts, spacer to organic handle and others.

Characteristics of surfactants are their molecular structure, determinedby NMR, chromatography (e.g., HPLC, GPC/SEC), FTIR, mass spectrometry,and titrations. Purity of surfactants is another characteristic examinedin addition to the fluorophile-hydrophile balance. A preferredembodiment includes oil-surfactant formulation for the application oflibrary emulsions is R-oil (HFE-7500) mixed with 2 wt % EA surfactant(“REAM”). Concentrations of EA or RR surfactant at 0.1 wt % or lower to5% or greater. Other formulations of oils and surfactants and othercomponents added to the aqueous phase are used to improved and optimizedthe performance of the droplets performance. Those properties of theoil-surfactant mixture include simple mixtures (i.e., one oil and onesurfactant, with varied surface concentration), co-surfactants, oilmixtures and additives, such as zonyl and TFA. Oil and surfactantmixture properties include surfactant solubility, critical micelleconcentration (CMC), surfactant diffusivity, and interfacial tension,i.e., dynamic and equilibrium. Emulsion properties are also accountedfor, those properties include size (absolute and size distribution),stability, transport, and biocompatibility. Stability relates directlyto the coalesced droplets and their deformability/breaking and shreddingability. More particularly, the stability of the droplets in theirgeneration, storage and shipping.

In general, production of surfactant and oils begins with the synthesisof surfactants and starting materials, such as PEG-diamine, EA and RRand also accounts for the purification processes, characterization,quality control, mixing and packaging. In one embodiment, thefluorosurfactant can be prepared by reacting the perfluorinatedpolyether DuPont Krytox 157 FSL, FSM, or FSH with aqueous ammoniumhydroxide in a volatile fluorinated solvent. The solvent and residualwater and ammonia can be removed with a rotary evaporator. Thesurfactant can then be dissolved (e.g., 2.5 wt %) in a fluorinated oil(e.g., Flourinert (3M)), which then serves as the continuous phase ofthe emulsion.

In another embodiment, a quaternary ammonium salt at the terminus of ahydrophilic oligomer is linked to a perfluoropolyether tail as shown inthe following formula:PFPE-C(0)NH—CH₂CH₂CH₂—(OCH₂CH₂)₃0-CH₂CH₂CH₂—N(CH₃)₃+I—. Some specificmolecular features of the present invention include, but are not limitedto, PFPE is from Krytox 157 FSH (Mn---6500), amide bond linking PFPE tohydrophile, propyl group immediately adjacent to the amide, propyl groupimmediately adjacent to the trimethylamino terminus. Specific structuralformations can alter performance relationships, for example, PFPE chainis sufficiently long for molecule to be soluble in perfluorinated oils,amide linker provides hydrolytic stability and hydrogen bonding site,and a combination of PEG and quaternary ammonium cation provide highanchoring strength to the aqueous phase as well as electrostaticrepulsion and steric hindrance to minimize reagent transport.

Variables in the molecular structure include, but are not limited to,PFPE molecular weight and polydispersity, PFPE structure, alternativeperfluorinated tail chemistries, PEG molecular weight andpolydispersity, shorter hydrocarbon linkers (ethyl or methyl versuspropyl), longer hydrocarbon spacers (C4 or higher), alternativecounterions, such as monovalent anions, monovalent, polyatomic anionsand di- or tri-valent counterions (to produce two or more tailfluorosurfactants). Further variables in the molecule structure includealternative linker chemistries (e.g., ether, ester, etc), alternativehydrophilic oligomers (e.g., polyalcohol, polyacrylamide, etc.),alternative quaternary ammonium cations, and alternative ionic groups(e.g., anionic terminus—carboxylate etc.; alternative cations).

The present invention is also directed to the coupling of PEG-diamineswith carboxylic acid terminated perfluoropolyether (Krytox 157) to formsurfactants. Specifically, the present invention is directed to afluorosurfactant molecule made by the ionic coupling of amine-terminatedpolyethyleneglycol (PEG-amine) with the carboxylic acid of DuPont Krytoxperfluoropolyether (PFPE). The resulting structure conveys goodperformance in the stabilization of aqueous droplets in fluorinated oilin a microfluidic system. Preferred surfactants are described in WO2008/021123. The present invention provides droplets with afluorosurfactant interface separating the aqueous droplet and itscontents from the surrounding immiscible fluorocarbon oil. In oneexample, DNA amplification reactions occurring inside these dropletsgenerate material that does not interact with the channel walls, andcollection of the DNA-containing droplets for subsequent manipulationand sequencing is straightforward. This technology provides a solutionfor amplification of DNA from single cells, allowing for both genotypingand whole genome amplification. In addition, use within a microfluidicdevice or platform as described herein achieves very high throughput,with droplets processed at rates in excess of 5000 droplets per second,enabling greater than 1×10⁶ single-cell droplets to be formed andmanipulated per hour.

Other examples of materials related to this invention include theformation of salts made by combination of various primary, secondary, ortertiary amines with PFPE carboxylic acid. The resulting amphiphilicstructure could be useful as a stand-alone surfactant or aco-surfactant. Similarly, fluorinated materials with carboxylic acidsother than Krytox PFPE could be used to form ionic fluorosurfactantswith various amine containing compounds.

Alternative amine-containing compounds for use with the presentinvention include, but are not limited to, PEG-monoamine (molecularweights range from 200 to 1,000,000 or more), PEG-diamine (molecularweights range from 200 to 1,000,000 or more), Multifunctional PEG amines(e.g., branched or dendritic structures), other hydrophilic polymersterminated with amines. Sugars include, but are not limited to, Sugars,Peptides, Biomolecules, Ethanolamine or Alkyl amines—primary, secondary,or tertiary (e.g., triethylamine, trimethylamine, methylamine,ethylamine, butylamine).

Alternative fluorinated groups for use with the present inventioninclude, but are not limited to, Krytox FSL and FSM (alternativemolecular weights), Demnum PFPE materials, Fluolink PFPE materials orFluorinated small molecules with carboxylic acids.

The data described herein show that the fluorosurfactants comprised ofPEG amine salts provide better performance (compared to otherfluorosurfactants) for stabilization of aqueous droplets in fluorinatedoils in droplet-based microfluidics applications. These novelsurfactants are useful either in combination with other surfactants oras a stand-alone surfactant system.

Driving Forces

The invention can use pressure drive flow control, e.g., utilizingvalves and pumps, to manipulate the flow of cells, particles, molecules,enzymes or reagents in one or more directions and/or into one or morechannels of a microfluidic device. However, other methods may also beused, alone or in combination with pumps and valves, such aselectro-osmotic flow control, electrophoresis and dielectrophoresis asdescribed in Fulwyer, Science 156, 910 (1974); Li and Harrison,Analytical Chemistry 69, 1564 (1997); Fiedler, et al. AnalyticalChemistry 70, 1909-1915 (1998) and U.S. Pat. No. 5,656,155. Applicationof these techniques according to the invention provides more rapid andaccurate devices and methods for analysis or sorting, for example,because the sorting occurs at or in a sorting module that can be placedat or immediately after a detection module. This provides a shorterdistance for molecules or cells to travel, they can move more rapidlyand with less turbulence, and can more readily be moved, examined, andsorted in single file, i.e., one at a time.

Positive displacement pressure or other positive pressure driven flow isa preferred way of controlling fluid flow and dielectrophoresis is apreferred way of manipulating droplets within that flow. The pressure atthe inlet module can also be regulated by adjusting the pressure on themain and sample inlet channels, for example, with pressurized syringesfeeding into those inlet channels. By controlling the pressuredifference between the oil and water sources at the inlet module, thesize and periodicity of the droplets generated may be regulated.Alternatively, a valve may be placed at or coincident to either theinlet module or the sample inlet channel connected thereto to controlthe flow of solution into the inlet module, thereby controlling the sizeand periodicity of the droplets. Periodicity and droplet volume may alsodepend on channel diameter, the viscosity of the fluids, and shearpressure. Examples of driving pressures and methods of modulating floware as described in WO 2006/040551; WO 2006/040554; WO 2004/002627; WO2004/091763; WO 2005/021151; WO 2006/096571; WO 2007/089541; WO2007/081385 and WO 2008/063227; U.S. Pat. No. 6,540,895 and U.S. PatentApplication Publication Nos. 20010029983 and 20050226742.

Droplet Inlet

The microfluidic device of the present invention includes one or moreinlet modules. An “inlet module” is an area of a microfluidic substratedevice that receives molecules, cells, small molecules or particles foradditional coalescence, detection and/or sorting. The inlet module cancontain one or more inlet channels, wells or reservoirs, openings, andother features which facilitate the entry of molecules, cells, smallmolecules or particles into the substrate. A substrate may contain morethan one inlet module if desired. Different sample inlet channels cancommunicate with the main channel at different inlet modules.Alternately, different sample inlet channels can communication with themain channel at the same inlet module. The inlet module is in fluidcommunication with the main channel. The inlet module generallycomprises a junction between the sample inlet channel and the mainchannel such that a solution of a sample (i.e., a fluid containing asample such as molecules, cells, small molecules (organic or inorganic)or particles) is introduced to the main channel and forms a plurality ofdroplets. The sample solution can be pressurized. The sample inletchannel can intersect the main channel such that the sample solution isintroduced into the main channel at an angle perpendicular to a streamof fluid passing through the main channel. For example, the sample inletchannel and main channel intercept at a T-shaped junction; i.e., suchthat the sample inlet channel is perpendicular (90 degrees) to the mainchannel. However, the sample inlet channel can intercept the mainchannel at any angle, and need not introduce the sample fluid to themain channel at an angle that is perpendicular to that flow. The anglebetween intersecting channels is in the range of from about 60 to about120 degrees. Particular exemplary angles are 45, 60, 90, and 120degrees.

Embodiments of the invention are also provided in which there are two ormore inlet modules introducing droplets of samples into the mainchannel. For example, a first inlet module may introduce droplets of afirst sample into a flow of fluid in the main channel and a second inletmodule may introduce droplets of a second sample into the flow of fluidin main channel, and so forth. The second inlet module is preferablydownstream from the first inlet module (e.g., about 30 Inn). The fluidsintroduced into the two or more different inlet modules can comprise thesame fluid or the same type of fluid (e.g., different aqueoussolutions). For example, droplets of an aqueous solution containing anenzyme are introduced into the main channel at the first inlet moduleand droplets of aqueous solution containing a substrate for the enzymeare introduced into the main channel at the second inlet module.Alternatively, the droplets introduced at the different inlet modulesmay be droplets of different fluids which may be compatible orincompatible. For example, the different droplets may be differentaqueous solutions, or droplets introduced at a first inlet module may bedroplets of one fluid (e.g., an aqueous solution) whereas dropletsintroduced at a second inlet module may be another fluid (e.g., alcoholor oil).

Droplet Interdigitation

Particular design embodiments of the microfluidic device describedherein allow for a more reproducible and controllable interdigitation ofdroplets of specific liquids followed by pair-wise coalescence of thesedroplets, described in further detail herein. The droplet pairs cancontain liquids of different compositions and/or volumes, which wouldthen combine to allow for a specific reaction to be investigated. Thepair of droplets can come from any of the following: (i) two continuousaqueous streams and an oil stream; (ii) a continuous aqueous stream, anemulsion stream, and an oil stream, or (iii) two emulsion streams and anoil stream. The term “interdigitation” as used herein means pairing ofdroplets from separate aqueous streams, or from two separate inletnozzles, for eventual coalescence.

Various nozzle designs enhance the interdigitation of droplets andfurther improves coalescence of droplets due to the better control ofthe interdigitation and smaller distance between pairs of droplets. Thegreater control over interdigitation allows for a perfect control overthe frequency of either of the droplets. To obtain the optimumoperation, the spacing between droplets and coupling of the droplets canbe adjusted by adjusting flow of any of the streams, viscosity of thestreams, nozzle design (including orifice diameter, the channel angle,and post-orifice neck of the nozzle). Examples of preferred nozzledesigns are as described in WO 2007/081385 and WO 2008/063227.

Droplet Coalescence or Combination

The microfluidic device of the present invention also includes one ormore coalescence modules. A “coalescence module” is within or coincidentwith at least a portion of the main channel at or downstream of theinlet module where molecules, cells, small molecules or particlescomprised within droplets are brought within proximity of other dropletscomprising molecules, cells, small molecules or particles and where thedroplets in proximity fuse, coalesce or combine their contents. Thecoalescence module can also include an apparatus, for generating anelectric force.

The electric force exerted on the fluidic droplet may be large enough tocause the droplet to move within the liquid. In some cases, the electricforce exerted on the fluidic droplet may be used to direct a desiredmotion of the droplet within the liquid, for example, to or within achannel or a microfluidic channel (e.g., as further described herein),etc.

The electric field can be generated from an electric field generator,i.e., a device or system able to create an electric field that can beapplied to the fluid. The electric field generator may produce an ACfield (i.e., one that varies periodically with respect to time, forexample, sinusoidally, sawtooth, square, etc.), a DC field (i.e., onethat is constant with respect to time), a pulsed field, etc. Theelectric field generator may be constructed and arranged to create anelectric field within a fluid contained within a channel or amicrofluidic channel. The electric field generator may be integral to orseparate from the fluidic system containing the channel or microfluidicchannel, according to some embodiments. As used herein, “integral” meansthat portions of the components integral to each other are joined insuch a way that the components cannot be in manually separated from eachother without cutting or breaking at least one of the components.

Techniques for producing a suitable electric field (which may be AC, DC,etc.) are known to those of ordinary skill in the art. For example, inone embodiment, an electric field is produced by applying voltage acrossa pair of electrodes, which may be positioned on or embedded within thefluidic system (for example, within a substrate defining the channel ormicrofluidic channel), and/or positioned proximate the fluid such thatat least a portion of the electric field interacts with the fluid. Theelectrodes can be fashioned from any suitable electrode material ormaterials known to those of ordinary skill in the art, including, butnot limited to, silver, gold, copper, carbon, platinum, copper,tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as wellas combinations thereof.

Preferred electrodes and patterned electrically conductive layers aredescribed in WO 2007/081385 and WO 2008/063227 and can be associatedwith any module of the device (inlet module, coalescence module, mixingmodule, delay module, detection module and sorting module) to generatedielectric or electric forces to manipulate and control the droplets andtheir contents.

Effective control of uncharged droplets within microfluidic devices canrequire the generation of extremely strong dielectric field gradients.The fringe fields from the edges of a parallel plate capacitor canprovide an excellent topology to form these gradients. The microfluidicdevice according to the present invention can include placing a fluidicchannel between two parallel electrodes, which can result in a steepelectric field gradient at the entrance to the electrodes due to edgeeffects at the ends of the electrode pair. Placing these pairs ofelectrodes at a symmetric channel split can allow precise bi-directionalcontrol of droplet within a device. Using the same principle, only withasymmetric splits, can allow single ended control of the dropletdirection in the same manner. Alternatively, a variation on thisgeometry will allow precise control of the droplet phase by shifting.

Dielectrophoresis is believed to produce movement of dielectric objects,which have no net charge, but have regions that are positively ornegatively charged in relation to each other. Alternating,non-homogeneous electric fields in the presence of droplets and/orparticles, such as cells or molecules, cause the droplets and/orparticles to become electrically polarized and thus to experiencedielectrophoretic forces. Depending on the dielectric polarizability ofthe particles and the suspending medium, dielectric particles will moveeither toward the regions of high field strength or low field strength.For example, the polarizability of living cells depends on theircomposition, morphology, and phenotype and is highly dependent on thefrequency of the applied electrical field. Thus, cells of differenttypes and in different physiological states generally possess distinctlydifferent dielectric properties, which may provide a basis for cellseparation, e.g., by differential dielectrophoretic forces.

Likewise, the polarizability of droplets also depends upon their size,shape and composition. For example, droplets that contain salts can bepolarized. According to formulas provided in Fiedler, et al. AnalyticalChemistry 70, 1909-1915 (1998), individual manipulation of singledroplets requires field differences (inhomogeneities) with dimensionsclose to the droplets.

The term “dielectrophoretic force gradient” means a dielectrophoreticforce is exerted on an object in an electric field provided that theobject has a different dielectric constant than the surrounding media.This force can either pull the object into the region of larger field orpush it out of the region of larger field. The force is attractive orrepulsive depending respectively on whether the object or thesurrounding media has the larger dielectric constant.

Manipulation is also dependent on permittivity (a dielectric property)of the droplets and/or particles with the suspending medium. Thus,polymer particles, living cells show negative dielectrophoresis athigh-field frequencies in water. For example, dielectrophoretic forcesexperienced by a latex sphere in a 0.5 MV/m field (10 V for a 20 micronelectrode gap) in water are predicted to be about 0.2 piconewtons (pN)for a 3.4 micron latex sphere to 15 pN for a 15 micron latex sphere(Fiedler, et al. Analytical Chemistry, 70, 1909-1915 (1998)). Thesevalues are mostly greater than the hydrodynamic forces experienced bythe sphere in a stream (about 0.3 pN for a 3.4 micron sphere and 1.5 pNfor a 15 micron sphere). Therefore, manipulation of individual cells orparticles can be accomplished in a streaming fluid, such as in a cellsorter device, using dielectrophoresis. Using conventional semiconductortechnologies, electrodes can be microfabricated onto a substrate tocontrol the force fields in a microfabricated sorting device of theinvention. Dielectrophoresis is particularly suitable for moving objectsthat are electrical conductors. The use of AC current is preferred, toprevent permanent alignment of ions. Megahertz frequencies are suitableto provide a net alignment, attractive force, and motion over relativelylong distances. See U.S. Pat. No. 5,454,472.

The electric field generator can be constructed and arranged (e.g.,positioned) to create an electric field applicable to the fluid of atleast about 0.01 V/micrometer, and, in some cases, at least about 0.03V/micrometer, at least about 0.05 V/micrometer, at least about 0.08V/micrometer, at least about 0.1 V/micrometer, at least about 0.3V/micrometer, at least about 0.5 V/micrometer, at least about 0.7V/micrometer, at least about 1 V/micrometer, at least about 1.2V/micrometer, at least about 1.4 V/micrometer, at least about 1.6V/micrometer, or at least about 2 V/micrometer. In some embodiments,even higher electric field intensities may be used, for example, atleast about 2 V/micrometer, at least about 3 V/micrometer, at leastabout 5 V/micrometer, at least about 7 V/micrometer, or at least about10 V/micrometer or more. As described, an electric field may be appliedto fluidic droplets to cause the droplets to experience an electricforce. The electric force exerted on the fluidic droplets may be, insome cases, at least about 10⁻¹⁶ N/micrometer³. In certain cases, theelectric force exerted on the fluidic droplets may be greater, e.g., atleast about 10⁻¹⁵ N/micrometer³, at least about 10⁻⁴ N/micrometer³, atleast about 10⁻¹³ N/micrometer³, at least about 10⁻¹² N/micrometer³, atleast about 10⁻¹, N/micrometer³, at least about 10⁻¹⁰ N/micrometer³, atleast about 10⁻⁹ N/micrometer³, at least about 10⁴ N/micrometer³, or atleast about 10⁻⁷ N/micrometer³ or more. The electric force exerted onthe fluidic droplets, relative to the surface area of the fluid, may beat least about 10⁻¹⁵N/micrometer², and in some cases, at least about10⁻¹⁴ N/micrometer², at least about 10⁻¹³ N/micrometer², at least about10⁻¹²N/micrometer², at least about 10^(−u)N/micrometer², at least about10^(−m)N/micrometer², at least about 10⁻⁹ N/micrometer², at least about10⁻⁸ N/micrometer², at least about le N/micrometer², or at least about10⁻⁶ N/micrometer² or more. In yet other embodiments, the electric forceexerted on the fluidic droplets may be at least about 10⁻⁹N, at leastabout 10⁴ N, at least about 10⁻⁷N, at least about 10⁻⁶N, at least about10⁻⁵N, or at least about 10⁻⁴ N or more in some cases.

Binding Agents, Solid Supports, and Washing

Methods of the invention involve use of first and second binding agents(also called ‘binders’) that can bind to the target analyte to form asandwich complex. The first and second binding agents have specificityfor different binding sites on the same target analyte, i.e., the firstand second binding agents bind different parts of the target analyte,where the analyte can be a single molecule or a stable complex ofmolecules. The binding agents may be any molecules that specificallybind to a target analyte in the sample. Exemplary binding agents includean antibody, an oligonucleotide, any protein based or nucleic acid basedbinding agent, or any agent capable of attaching to target analytes.Further binding agents embodied in methods of the invention include DNA,RNA, LNA (locked nucleic acids), PNA (peptide nucleic acid), a ligand,an irreversible inhibiting small molecule, a metabolite, a lipid, asugar, a synthetic polymer, or other non-peptide or nucleic acid-basedbinding agent, or combinations of the above.

In certain embodiments, the binding agent is an antibody. Generalmethodologies for antibody production, including criteria to beconsidered when choosing an animal for the production of antisera, aredescribed in Harlow et al. (Antibodies, Cold Spring Harbor Laboratory,pp. 93-117, 1988). For example, an animal of suitable size such asgoats, dogs, sheep, mice, or camels are immunized by administration ofan amount of immunogen effective to produce an immune response. Anexemplary protocol is as follows. The animal is injected with 100milligrams of antigen resuspended in adjuvant, for example Freund'scomplete adjuvant, dependent on the size of the animal, followed threeweeks later with a subcutaneous injection of 100 micrograms to 100milligrams of immunogen with adjuvant dependent on the size of theanimal, for example Freund's incomplete adjuvant. Additionalsubcutaneous or intraperitoneal injections every two weeks withadjuvant, for example Freund's incomplete adjuvant, are administereduntil a suitable titer of antibody in the animal's blood is achieved.Exemplary titers include a titer of at least about 1:5000 or a titer of1:100,000 or more, i.e., the dilution having a detectable activity. Theantibodies are purified, for example, by affinity purification oncolumns containing protein G resin or target-specific affinity resin.

The technique of in vitro immunization of human lymphocytes is used togenerate monoclonal antibodies. Techniques for in vitro immunization ofhuman lymphocytes are well known to those skilled in the art. See, e.g.,Inai, et al., Histochemistry, 99(5):335 362, May 1993; Mulder, et al.,Hum. Immunol., 36(3):186 192, 1993; Harada, et al., J. Oral Pathol.Med., 22(4):145 152, 1993; Stauber, et al., J. Immunol. Methods,161(2):157 168, 1993; and Venkateswaran, et al., Hybridoma, 11(6) 729739, 1992. These techniques can be used to produce antigen-reactivemonoclonal antibodies, including antigen-specific IgG, and IgMmonoclonal antibodies.

In certain embodiments, the binding agent is an aptamer. As used herein,“aptamer” and “nucleic acid ligand” are used interchangeably to refer toa nucleic acid that has a specific binding affinity for a targetmolecule, such as a protein. Like all nucleic acids, a particularnucleic acid ligand may be described by a linear sequence of nucleotides(A, U, T, C and G), typically 15-40 nucleotides long. Nucleic acidligands can be engineered to encode for the complementary sequence of atarget protein known to associate with the presence or absence of aspecific disease.

In solution, the chain of nucleotides form intramolecular interactionsthat fold the molecule into a complex three-dimensional shape. The shapeof the nucleic acid ligand allows it to bind tightly against the surfaceof its target molecule. In addition to exhibiting remarkablespecificity, nucleic acid ligands generally bind their targets with veryhigh affinity, e.g., the majority of anti-protein nucleic acid ligandshave equilibrium dissociation constants in the picomolar to lownanomolar range.

Aptamers used in the compositions of the invention depend upon thetarget tissue. Nucleic acid ligands may be discovered by any methodknown in the art. In one embodiment, nucleic acid ligands are discoveredusing an in vitro selection process referred to as SELEX (SystematicEvolution of Ligands by Exponential enrichment). See for example Gold etal. (U.S. Pat. Nos. 5,270,163 and 5,475,096), the contents of each ofwhich are herein incorporated by reference in their entirety. SELEX isan iterative process used to identify a nucleic acid ligand to a chosenmolecular target from a large pool of nucleic acids. The process relieson standard molecular biological techniques, using multiple rounds ofselection, partitioning, and amplification of nucleic acid ligands toresolve the nucleic acid ligands with the highest affinity for a targetmolecule. The SELEX method encompasses the identification ofhigh-affinity nucleic acid ligands containing modified nucleotidesconferring improved characteristics on the ligand, such as improved invivo stability or improved delivery characteristics. Examples of suchmodifications include chemical substitutions at the ribose and/orphosphate and/or base positions. There have been numerous improvementsto the basic SELEX method, any of which may be used to discover nucleicacid ligands for use in methods of the invention.

In certain embodiments, the binding agent is an oligonucleotide. Methodsof synthesizing oligonucleotides are known in the art. See, e.g.,Sambrook et al. (DNA microarray: A Molecular Cloning Manual, Cold SpringHarbor, N.Y., 2003) or Maniatis, et al. (Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y., 1982), the contents of each of whichare incorporated by reference herein in their entirety. Suitable methodsfor synthesizing oligonucleotide probes are also described in Caruthers(Science 230:281-285, 1985), the contents of which are incorporated byreference. Oligonucleotides can also be obtained from commercial sourcessuch as Operon Technologies, Amersham Pharmacia Biotech, Sigma, and LifeTechnologies. The oligonucleotides can have an identical meltingtemperature. The lengths of the probes can be extended or shortened atthe 5′ end or the 3′ end to produce oligonucleotides with desiredmelting temperatures. Also, the annealing position of eacholigonucleotide can be designed such that the sequence and length of theprobe yield the desired melting temperature. The simplest equation fordetermining the melting temperature of probes smaller than 25 base pairsis the Wallace Rule (Td=2(A+T)+4(G+C)). Computer programs can also beused to design oligonucleotides, including but not limited to ArrayDesigner Software (Arrayit Inc.), Oligonucleotide Probe Sequence DesignSoftware for Genetic Analysis (Olympus Optical Co.), NetPrimer, andDNAsis from Hitachi Software Engineering. The TM (melting temperature)of each probe is calculated using software programs such as OligoDesign, available from Invitrogen Corp.

In certain embodiments, the oligonucleotides can include two portions, aportion that includes a nucleotide sequence with substantialcomplementarity to a target analyte, so that the oligonucleotidehybridizes with the target analyte. The oligonucleotidescan also includea universal region, i.e., a synthetic sequence that is not found in thegenome of the organism of interest and that is identical in all of theoligonucleotides. The universal sequence may be a homopolymer, e.g.,poly(A) or may be a sequence composed of many different bases. Theuniversal region of the oligonucleotides is useful as a primer site forconducting secondary enzymatic reactions to link any number of sequencebased or chemical moieties that are relevant to downstream processingand analysis.

Oligonucleotides suitable for use in the present invention include thoseformed from nucleic acids, such as RNA and/or DNA, nucleic acid analogs,locked nucleic acids, modified nucleic acids, and chimeric probes of amixed class including a nucleic acid with another organic component suchas peptide nucleic acids. Exemplary nucleotide analogs include phosphateesters of deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine,adenosine, cytidine, guanosine, and uridine. Other examples ofnon-natural nucleotides include a xanthine or hypoxanthine;5-bromouracil, 2-aminopurine, deoxyinosine, or methylated cytosine, suchas 5-methylcytosine, and N4-methoxydeoxycytosine. Also included arebases of polynucleotide mimetics, such as methylated nucleic acids,e.g., 2′-O-methRNA, peptide nucleic acids, modified peptide nucleicacids, and any other structural moiety that can act substantially like anucleotide or base, for example, by exhibiting base-complementarity withone or more bases that occur in DNA or RNA. Also included arenucleotides modified for use in photo-activated ligation or cleavage,e.g. 4-thiothymidine,1-[2-Nitro-5-(6-trifluoroacetylcaproamidomethyl)phenyl]-ethyl-[2-cyano-ethyl-(N,N-diisopropyl)]-phosphoramidite,5-carboxyvinyl-2′-deoxyuridine.

The length of the nucleic acid binding agents are not critical, as longas they are capable of specific binding to the target regions. In fact,oligonucleotides may be of any length. For example, oligonucleotides maybe as few as 5 nucleotides, or as much as 5000 nucleotides. Exemplaryoligonucleotides are 5-mers, 10-mers, 15-mers, 20-mers, 25-mers,50-mers, 100-mers, 200-mers, 500-mers, 1000-mers, 3000-mers, or5000-mers. Methods for determining an optimal oligonucleotides lengthare known in the art. See, e.g., Shuber (U.S. Pat. No. 5,888,778). Thefirst and second binding agents do not have to be of the same length. Incertain embodiments, the first and second binding agents are the samelength, while in other embodiments, the first and second binding agentsare of different lengths.

In certain embodiments, a competitive inhibitor is introduced to thedroplet library or the sample in order to enhance the specificity of theisolated sandwich formation. In some embodiments, the sample containstarget analytes and unspecific analytes similar to the target analyteswhich may result in a false positive connection between the bindingagent specific to the target analyte and the unspecific analyte. If afalse positive connection is made, the representative value of theidentifiers to the target analyte reduces in specificity. In oneembodiment unspecific nuclei acid analytes may contain a sequence with asingle base difference in a sequence. Unspecific analytes include anydifference from a target protein or nucleic acid or other target analytethat would result in an affinity of the target analyte identifier to theunspecific analyte. FIG. 7 depicts a non-limiting embodiment of the useof a competitive inhibitor to prevent a binding agent with an identifierand, or a capture molecule from binding to a competitive unspecificanalyte, wild type (C) allele, within the sample, when the targetanalyte is a rare mutant (G) allele. In the FIG. 7 embodiment, thecompetitive inhibitor includes a minor groove binder motif thatincreases the affinity to the wild type unspecific analyte, and binds tothe unspecific analyte prior to the target analyte specific bindingagent, or competes the target analyte specific binding agent off.Methods of the invention provide for the competitive inhibitor to beDNA, RNA, PNA, and LNA, and any other nucleic acid, or protein, or otherbinding agents capable of preventing false positive attachment to aunspecific analyte. The competitive molecules can be included in thesample, the droplet library, or both.

Generally, the second binding agent (also called the ‘capture-taggedbinder’) is configured such that it can be coupled to a solid support ineither a reversible or irreversible manner. For example, a terminalportion of the second binding agent may be functionalized with aterminal amine such that it can covalently bind an epoxide coatedsurface. The terminal amine that can form a covalent bond with anepoxide coated bead. In this embodiment, the epoxide coated bead isintroduced to the binding agent now bearing an amine group. Thehighly-reactive epoxide ring opens, and a reactive carbon binds to theamine group on the copy. Further description of amine attachment isshown for example in Harris et al. (U.S. Pat. No. 7,635,562; Joos etal., Analytical Biochemistry 247:96-101, 1997; Oroskar et al., Clin.Chem. 42:1547-1555, 1996; and Khandjian, Mol. Bio. Rep. 11:107-115,1986, the content of each of which is incorporated by reference hereinin its entirety.

Alternatively, a part of the second binding agent is functionalized withone member of a binding pair while a surface of the solid support isfunctionalized with the other member of the binding pair (e.g.,biotin/avidin; biotin/streptavidin/ or digoxigenin/anti-digoxigenin).The second binding agent, now functionalized with a member of thebinding pair (i.e., member of a capture tag pair) is brought intoproximity of the solid support coated with the other member of thebinding pairs. The two members of the binding pair interact toimmobilize the complexes onto the solid support. See Harris et al. U.S.Pat. No. 7,635,562; Taylor et al., J. Phys. D. Appl. Phys. 24:1443,1991); and Smith et al., Science 253:1122, 1992, the content of each ofwhich is incorporated by reference herein in its entirety. Additionalstringency can be provided through using tandem binding motif pairs.

Exemplary couplings include but are not limited to 1) a biotin capturetag for use with a streptavidin, avidin, or alternative modifiedderivatives of streptavidin or avidin solid supports; 2) a desthiobiotincapture tag for use with a streptavidin, avidin, or alternative modifiedderivatives of streptavidin or avidin solid support; 3) a antigencapture tag including FLAG, hemagglutinin, calmodulin-binding domain,histadinemultimers, or other common epitope tags for use with theircognate binding partners including anti-FLAG, anti-hemagglutinin,anti-calmodulin as the solid support. Further couplings embodied in theinvention include metal-binding domains for binding histidine-tags, orbinders targeting other binders e.g. antibodies bound to otherantibodies or antibody-binders like ProteinA/G.

In certain non-limiting embodiments, various formats are used to attachthe capture tags to a bead, or other solid support. Several embodimentsfor coupling include, but are not limited to 1) the capture tag attachesto beads present in the droplet library; 2) the capture tag attaches tobeads present in sample droplets; 3) the capture tag attaches to beadsafter release of the combined droplet contents; and 4) the capture tagattaches on a well plate after release of the combined droplet contents.

In some embodiments of the invention, the target analyte sandwich isisolated from the unbound sample components without the use of capturetags or immobilization to a stable support. Such embodiments include butare not limited to using magnetic energy, chemical reactions,pressurized separation with shear stress, or any other system capable ofisolating target analyte sandwiches with specificity to allow foranalyzing only the target analyte. The preferred embodiment of theinvention utilizes capture molecules having capture tags for binding toa stable support.

The support may be a bead that is present in the droplet or it may be asupport outside of the droplet. Supports for use in the invention can betwo- or three-dimensional and can comprise a planar surface (e.g., aglass slide) or can be shaped. A support substrate can include glass(e.g., controlled pore glass (CPG)), quartz, plastic (such aspolystyrene (low cross-linked and high cross-linked polystyrene),polycarbonate, polypropylene and poly(methymethacrylate)), acryliccopolymer, polyamide, silicon, metal (e.g., alkanethiolate-derivatizedgold), cellulose, nylon, latex, dextran, gel matrix (e.g., silica gel),polyacrolein, composites, or other materials.

Suitable three-dimensional supports include, for example, spheres,microparticles, beads, membranes, slides, plates, micromachined chips,tubes (e.g., capillary tubes), microwells, microfluidic devices,channels, filters, or any other structure suitable for anchoring thecapture-tagged binder. Supports can include planar arrays or matricescapable of having regions that include populations of nucleic acids,peptides, sugars, or other molecules. Examples includenucleoside-derivatized CPG and polystyrene slides; derivatized magneticslides; polystyrene grafted with polyethylene glycol, and the like.

Once the immobilization step occurs such that the sandwich is bound tothe solid support, a wash step is performed to isolate sandwichcomplexes bound to the solid support from remaining components in thesample. In one embodiment, a wash buffer may have sufficient stringencyto remove the unincorporated binders and unincorporated non-specificanalytes without disrupting the sandwich. In certain embodiments, thewash may just impose a shear stress to remove unwanted and unattachedsample and binding elements, however a wash containing binding elementsspecific to the unwanted sample may increase specificity in the targetanalyte analysis.

Target Identifier and Sample Identifier Molecules

Generally, the first binding agent, also called the ‘barcoded binder’,includes an identifier molecule, i.e., a target identifier. The targetidentifier molecule may be any molecule that is differentiallydetectable by any detection techniques known in the art. Exemplarydetection methods include radioactive detection, optical absorbancedetection, e.g., UV-visible absorbance detection, optical emissiondetection, e.g., fluorescence; phosphorescence or chemiluminescence;Raman scattering, magnetic detection, or mass spectral detection. Incertain embodiments, the identifier is an optically detectable label,such as a fluorescent label. Examples of fluorescent labels include, butare not limited to, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine and derivatives: acridine, acridineisothiocyanate; 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid(EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY;Brilliant Yellow; coumarin and derivatives; coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron.™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′ tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Atto dyes, Cy3; Cy5; Cy5.5; Cy7; IRD 700;IRD 800; La Jolta Blue; phthalo cyanine; and naphthalo cyanine.Preferred fluorescent labels are cyanine-3 and cyanine-5. Labels otherthan fluorescent labels are contemplated by the invention, includingother optically-detectable labels.

Fluorescently labeled nucleotides may be produced by various techniques,such as those described in Kambara et al. (Bio/Technol., 6:816-21,1988); Smith et al. (Nucl. Acid Res., 13:2399-2412, 1985); and Smith etal. (Nature, 321: 674-679, 1986). The fluorescent dye may be linked tothe deoxyribose by a linker arm that is easily cleaved by chemical orenzymatic means. There are numerous linkers and methods for attachinglabels to nucleotides, as shown in Oligonucleotides and Analogues: APractical Approach (IRL Press, Oxford, 1991); Zuckerman et al.(Polynucleotides Res., 15: 5305-5321, 1987); Sharma et al.(Polynucleotides Res., 19:3019, 1991); Giusti et al. (PCR Methods andApplications, 2:223-227, 1993); Fung et al. (U.S. Pat. No. 4,757,141);Stabinsky (U.S. Pat. No. 4,739,044); Agrawal et al. (TetrahedronLetters, 31:1543-1546, 1990); Sproat et al. (Polynucleotides Res.,15:4837, 1987); and Nelson et al. (Polynucleotides Res., 17:7187-7194,1989). Extensive guidance exists in the literature for derivatizingfluorophore and quencher molecules for covalent attachment via commonreactive groups that may be added to a nucleotide. Many linking moietiesand methods for attaching fluorophore moieties to nucleotides alsoexist, as described in Oligonucleotides and Analogues, supra; Guisti etal., supra; Agrawal et al, supra; and Sproat et al., supra.

In other embodiments, the target identifier is a sequence ofoligonucleotides that constitutes a unique identifier, or barcode.Attaching barcode sequences to other molecules, such as nucleic acids,is shown for example in Kahvejian et al. (U.S. patent application number2008/0081330), and Steinman et al. (International patent applicationnumber PCT/US09/64001), the content of each of which is incorporated byreference herein in its entirety. Methods for designing sets of barcodesequences and other methods for attaching barcode sequences are shown inU.S. Pat. Nos. 6,138,077; 6,352,828; 5,636,400; 6,172,214; 6,235,475;7,393,665; 7,544,473; 5,846,719; 5,695,934; 5,604,097; 6,150,516;RE39,793; 7,537,897; 6,172,218; and 5,863,722, the content of each ofwhich is incorporated by reference herein in its entirety. In certainembodiments, a single barcode is attached to each identifier. In otherembodiments, a plurality of barcodes, e.g., two barcodes, are attachedto each identifier.

In certain embodiments, the barcode identifier can include features thatmake it useful in nucleic acid sequencing reactions. For example thebarcode sequences are designed to have minimal or no homopolymerregions, i.e., 2 or more of the same base in a row such as AA or CCC,within the barcode sequence.

Methods of designing sets of nucleic acid barcode sequences is shown forexample in Brenner et al. (U.S. Pat. No. 6,235,475), the contents ofwhich are incorporated by reference herein in their entirety. In certainnon-limiting embodiments, the barcode sequences range from about 4nucleotides to about 25 nucleotides. In a particular embodiment, thebarcode sequences range from about 4 nucleotides to about 7 nucleotides.

When the first binding agent is a nucleic acid, the barcode sequence canbe attached to the nucleic acid with an enzyme, or the entire nucleicacid can be synthesised. The enzyme may be a ligase or a polymerase. Theligase may be any enzyme capable of ligating an oligonucleotide (RNA orDNA) to the nucleic acid molecule. Suitable ligases include T4 DNAligase and T4 RNA ligase (such ligases are available commercially, fromNew England Biolabs). Methods for using ligases are well known in theart. The polymerase may be any enzyme capable of adding nucleotides tothe 3′ and the 5′ terminus of template nucleic acid molecules.Photo-ligation, chemical attachment, or other methods may also be usedto attach the barcode sequence to the nucleic acid-based binder.Non-covalent attachment methods may also be used. One embodiment useshybridization of complimentary oligonucleotides, between a nucleic acidcovalently attached to the first binding agent and an oligonucleotidecontaining a target identifier.

The target identifier may be coupled to the first binding agent in areleasable manner, such that the identifier may be separated from thefirst binding agent for purposes of detection in certain embodiments.Alternatively, the target identifier is irreversibly coupled to thefirst binding agent and detection of the identifier occurs on thecomplex with the analyte.

In one embodiment for attachment, a modified terminal oligonucleotide isincorporated to the end of an oligonucleotide-encoded identifier. Theincorporation may include attaching a UV photo-cleavable primary aminogroup onto the 5′ end of the oligonucleotide binding agent, andsubsequently directly attaching the identifier to a protein basedbinding agent via the amino group. In another embodiment, a UVphoto-cleavable oligonucleotide is incorporated into a nucleic acidbased binding agent.

Further attachment strategies involve using a bi-functionalcross-linking reagent to directly attach an amino acid containingbinding agent to an oligonucleotide-based identifier. Embodiments of themethod include indirect method attachments, including but not limitedto, hybridizing, or annealing, an oligonucleotide identifier to acomplimentary oligonucleotide that is either linked to a protein-basedbinding agent, or incorporated in the sequence of a nucleic acid-basedbinding agents. Other formats or combinations of the above may also beincluded, such as attaching a biotinylated or desthiotinylated barcodedoligonucleotide bound to a streptavidin-modified binding agent;attaching DIG, dye, or biotinylated identifying oligonucleotide bound toan antibody to the same motifs bound to the binding agent; and otherdimerization motifs, including protein-based nucleic acid-based, orchemical based. Other attachment strategies may be used for protein,nucleic acid, or non-protein or non-nucleic acid binding agents (e.g.chemical modification of lipids, sugars, or synthetic small molecules).

Methods of the invention provide for releasing identifiers isolatedafter the unbound sample components are removed in the wash. Theidentifier may be released from the target analyte sandwich using avariety of methods including but not limited to 1) Light-inducedrelease, for example UV-induced photocleavage; 2) Enzymatic release, forexample restriction endonuclease, exonuclease, protease; 3) Chemicalinduced release, for example. metal-catalyzed oxidation; 4)Temperature-induced release, for example release of annealedoligonucleotide; and 5) combinations of the above releasing methods.

Methods of the invention provide for a capture-tagged binding moleculeto correspond with the binding agent having the target identifier, asthey are co-encapsulated first in the droplet library element, andsubsequently after combination with a sample droplet. The capturemolecule and binding agent are considered partner pairs because both arespecific to a target analyte and will create a sandwich assay in thepresence of the analyte. The barcode identifiers are designed such thateach barcode is directly correlated to a particular set of bindingagents that are placed together in a single droplet library element forbinding a particular target analyte. This allows barcode reads to becorrelated back to the binding agents, and thus the target analyte forquantification. Examples of this encoding strategy are shown in FIGS. 5and 6. In FIG. 5, A) Two binding reagents types are constructed:Barcoded Binders and Capture-Tag Binders; B) Pairs of target-specificbinders are made into a droplet library, with each set of target bindersin separate droplets; C) The sample is made into sample droplets, and D)combined with the library droplets to initiate highly parallel‘single-plex’ binding reactions. After binding is complete, productivesandwiches are E) captured via the capture-tag (streptavidin (SA) biotin(B) interaction shown), and washed to remove unbound material; F) Thecaptured barcodes are released, recovered, and processed for reading; G)Reads for each barcode are counted (e.g. using sequencing). In FIG. 6,A) A binder pair targeting two different regions of the same analyteenable counting single target analytes; B&C) Binder pairs targetingdifferent analytes in a complex enable identification and digitalquantification of analyte complexes; D) A binder pair targeting twodifferent regions of the same analyte, with one target being a specificmodification (e.g. protein post-translational); E) Cross-linked orstable complexes can be analyzed (e.g. protein-nucleic acid); F-J)Identification and counting of various nucleic acid molecules and motifsare shown (detailed descriptions in the text). Note that the BinderBarcode information includes details on which binders are in the librarydroplet (e.g. “3:1:2” in example C means Binder3 in the same droplet asBinder1 and Capture-Tag Binder2)

In some embodiments of the invention, the capture molecule may have itsown or similar identifier to the corresponding identifier attached tothe binding agent to use for quantification. In one embodiment, thecontrol identifiers are read after a separate release step.

Another method of the invention provides for creating a sampleidentifier to provide more coding information. In one embodiment, thesample identifier contains information towards identifying that thetarget analyte came from a certain sample, but sample identifierinformation may be any extra information useful in analyzing the targetanalyte. The sample identifier is linked to the target analyteidentifier, when the sample and library droplet are combined. Oneembodiment includes a sample identifier and an identifier linkingcomponent within a sample droplet, or the sample linking component canbe introduced along with the library droplet, or added in a subsequentstep (e.g. another combination or addition to the combined sample andlibrary droplet). In one embodiment, the sample droplet is merged with alibrary droplet containing a first binding agent having a target analyteidentifier and a capture-tagged molecule. When the droplets are merged,a sandwich of the target analyte, first binding agent, andcapture-tagged molecule is formed and the sample identifier litigateswith the target analyte identifier. In certain embodiments theidentifier linking component is a stabilizing molecule to facilitate theligation of the sample identifier and the target analyte identifier. Incertain embodiments, the identifier linking template is not needed, andlitigation of the identifier occurs, for example, from affinity of oneidentifier to the second identifier followed by a reactive step (e.g.ligase, chemical catalysis, light-induced). The unattached samplecomponents are then removed leaving only the isolated sandwich. Thecombined sample and target analyte identifier are then used to analyze,identify and quantify the target analyte. FIG. 8 demonstrates anembodiment of the invention for creating sandwich assays in combinationwith combining a sample barcode identifier to a target analyte barcodeidentifier. In FIG. 8A) Sample Barcode and the Barcode Linking Templateare added to bulk Sample A, then made into droplets; B) Sample andLibrary droplets are combined and incubated to form a sandwich in thepresence of the analyte. The Binder Barcode is coupled to the SampleBarcode (e.g. Binder Barcode terminal modified base (*) is photo-ligatedto the 5-prime terminal modified base (*) on the Sample Barcode); D) Thedroplet contents are released and the full sandwich on the capturesurface is washed. The Combined Barcode is released (e.g.photo-release); E) Reads for each barcode are counted (e.g. usingsequencing). FIG. 9 depicts two embodiments of the invention wherein theBinder Barcode and the Sample Barcode are combined into one barcode foranaylsis. In A, modified oligonucleotides are present in the 5-primeterminus of the Sample Barcode and the 3-prime terminus of the BinderBarcode, such that when they are brought together by the CouplingComponent (here a oligonucleotide template) and irradiated with 366 nmwavelength light, a photo-catalyzed reaction forms a covalent coupling(e.g. here using 4-thiothymidine (T*)), thus resulting in a combinedsample and target analyte identifier. The Coupling Component is meltedoff, and the final identifier can be released by a photo-cleavage method(using a shorter wavelength). In FIG. 9B, a ligase is used to combinethe two barcodes, with the Coupling Component template to align,followed by release using a restriction enzyme site. In B1) The BarcodeBinder, the Sample Barcode and the Barcode Coupling Component (e.g.template spanning the two barcodes) are in the same droplet and; B2)bind as a complex during incubation. The Barcode Coupling Component is anucleic acid template that hybridizes to both the 5-prime and 3-primeend of the Binder Barcode (forming a hairpin) and the 5-prime end of theSample Barcode; 2C) The 3-prime end of the Binder Barcode is ligatedonto the 5-prime end of the Sample Barcode using a ligase; 2D) Theannealed 5-prime end of the ligated Combined Barcode encodes arestriction endonuclease site that can be cleaved for release of theCombined Barcode.

Another method of the invention provides for a sample identifier toprovide more unique coding information. The sample identifier isintroduced into the assay to ligate to the target analyte identifier toprovide layered information about the target analyte, for example thetarget analyte is X-protein and X-protein came from sample-Y. In oneembodiment, the sample identifier contains identifying information thatthe target analyte came from a certain sample. The sample identifier,however, may be coded to contain any extra information useful inanalyzing the target analyte.

Detection, Reading and Counting Identifiers

Any detection method can be used that is suitable for the type of labelemployed. Thus, exemplary detection methods include radioactivedetection, optical absorbance detection, e.g., UV-visible absorbancedetection, optical emission detection, e.g., fluorescence,chemiluminescence, or phosphorescence, Raman scattering, magneticdetection, or mass spectral detection. For fluorescence labeling, afluorescence microscope apparatus, such as described in Fodor (U.S. Pat.No. 5,445,934) and Mathies et al. (U.S. Pat. No. 5,091,652), may be usedto detect the identifiers. Devices capable of sensing fluorescence froma single molecule include scanning tunneling microscope (siM) and theatomic force microscope (AFM). Hybridization patterns may also bescanned using a CCD camera (e.g., Model TE/CCD512SF, PrincetonInstruments, Trenton, N.J.) with suitable optics (Ploem, in Fluorescentand Luminescent Probes for Biological Activity Mason, T. G. Ed.,Academic Press, Landon, pp. 1-11 (1993), such as described in Yershov etal., Proc. Natl. Acad. Sci. 93:4913 (1996), or may be imaged by TVmonitoring. For radioactive signals, a phosphorimager device can be used(Johnston et al., Electrophoresis, 13:566, 1990; Drmanac et al.,Electrophoresis, 13:566, 1992; 1993). Other commercial suppliers ofimaging instruments include General Scanning Inc., (Watertown, Mass. onthe World Wide Web at genscan.com), Genix Technologies (Waterloo,Ontario, Canada; on the World Wide Web at confocal.com), and AppliedPrecision Inc. A number of approaches can be used to detect theidentifiers. Optical setups include near-field scanning microscopy,far-field confocal microscopy, wide-field epi-illumination, lightscattering, dark field microscopy, photoconversion, single and/ormultiphoton excitation, spectral wavelength discrimination, fluorophoridentification, evanescent wave illumination, and total internalreflection fluorescence (TIRF) microscopy. In general, certain methodsinvolve detection of laser-activated fluorescence using a microscopeequipped with a camera. Suitable photon detection systems include, butare not limited to, photodiodes and intensified CCD cameras. Forexample, an intensified charge couple device (ICCD) camera can be used.The use of an ICCD camera to image individual fluorescent dye moleculesin a fluid near a surface provides numerous advantages. For example,with an ICCD optical setup, it is possible to acquire a sequence ofimages (movies) of fluorophores.

Some embodiments of the present invention use TIRF microscopy forimaging. TIRF microscopy uses totally internally reflected excitationlight and is well known in the art. See, e.g., the World Wide Web atnikon-instruments .jp/eng/page/products/tirf.aspx. In certainembodiments, detection is carried out using evanescent wave illuminationand total internal reflection fluorescence microscopy. An evanescentlight field can be set up at the surface, for example, to imagefluorescently-labeled nucleic acid molecules. When a laser beam istotally reflected at the interface between a liquid and a solidsubstrate (e.g., a glass), the excitation light beam penetrates only ashort distance into the liquid. The optical field does not end abruptlyat the reflective interface, but its intensity falls off exponentiallywith distance. This surface electromagnetic field, called the“evanescent wave”, can selectively excite fluorescent molecules in theliquid near the interface. The thin evanescent optical field at theinterface provides low background and facilitates the detection ofsingle molecules with high signal-to-noise ratio at visible wavelengths.

The evanescent field also can image fluorescently-labeled nucleotidesupon their incorporation into the attached template/primer complex inthe presence of a polymerase. Total internal reflectance fluorescencemicroscopy is then used to visualize the attached template/primer duplexand/or the incorporated nucleotides with single molecule resolution.

According to some embodiments of the invention, after barcoded sandwichcomplexes have been isolated from the remaining components of thesample, the barcode sequences are released from the first bindingagents. In one embodiment, the barcode sequences are joined to eachother to produce a single contiguous molecule containing multiplebarcodes in series. The individual barcodes, collections of separatebarcodes, or individual or collections of multiple barcodes connected inseries or other arrangements can be detected with or withoutamplification. In one embodiment, the barcode or barcode collections aresubjected to an amplification reaction (e.g., PCR or rolling circleamplification) to produce multiple linear copies (concatamers), linkedend-to-end. The amplification products are then sequenced.

Sequencing may be by any method known in the art. DNA sequencingtechniques include classic dideoxy sequencing reactions (Sanger method)using labeled terminators or primers and gel separation in slab orcapillary, sequencing by synthesis using reversibly terminated labelednucleotides, pyrosequencing, 454 sequencing, allele specifichybridization to a library of labeled oligonucleotide probes, sequencingby synthesis using allele specific hybridization to a library of labeledclones that is followed by ligation, real time monitoring of theincorporation of labeled nucleotides during a polymerization step,polony sequencing, and SOLiD sequencing. Sequencing of separatedmolecules has more recently been demonstrated by sequential or singleextension reactions using polymerases or ligases as well as by single orsequential differential hybridizations with libraries of probes.

A sequencing technique that can be used in the methods of the providedinvention includes, for example, Helicos True Single Molecule Sequencing(tSMS) (Harris T. D. et al. (2008) Science 320:106-109). In the tSMStechnique, a DNA sample is cleaved into strands of approximately 100 to200 nucleotides, and a polyA sequence is added to the 3′ end of each DNAstrand. Each strand is labeled by the addition of a fluorescentlylabeled adenosine nucleotide. The DNA strands are then hybridized to aflow cell, which contains millions of oligo-T capture sites that areimmobilized to the flow cell surface. The templates can be at a densityof about 100 million templates/cm². The flow cell is then loaded into aninstrument, e.g., HeliScope.™ sequencer, and a laser illuminates thesurface of the flow cell, revealing the position of each template. A CCDcamera can map the position of the templates on the flow cell surface.The template fluorescent label is then cleaved and washed away. Thesequencing reaction begins by introducing a DNA polymerase and afluorescently labeled nucleotide. The oligo-T nucleic acid serves as aprimer. The polymerase incorporates the labeled nucleotides to theprimer in a template directed manner. The polymerase and unincorporatednucleotides are removed. The templates that have directed incorporationof the fluorescently labeled nucleotide are detected by imaging the flowcell surface. After imaging, a cleavage step removes the fluorescentlabel, and the process is repeated with other fluorescently labelednucleotides until the desired read length is achieved. Sequenceinformation is collected with each nucleotide addition step. Furtherdescription of tSMS is shown for example in Lapidus et al. (U.S. Pat.No. 7,169,560), Lapidus et al. (U.S. patent application number2009/0191565), Quake et al. (U.S. Pat. No. 6,818,395), Harris (U.S. Pat.No. 7,282,337), Quake et al. (U.S. patent application number2002/0164629), and Braslaysky, et al., PNAS (USA), 100: 3960-3964(2003), the contents of each of these references is incorporated byreference herein in its entirety.

Another example of a DNA sequencing technique that can be used in themethods of the provided invention is 454 sequencing (Roche) (Margulies,M et al. 2005, Nature, 437, 376-380). 454 sequencing involves two steps.In the first step, DNA is sheared into fragments of approximately300-800 base pairs, and the fragments are blunt ended. Oligonucleotideadaptors are then ligated to the ends of the fragments. The adaptorsserve as primers for amplification and sequencing of the fragments. Thefragments can be attached to DNA capture beads, e.g.,streptavidin-coated beads using, e.g., Adaptor B, which contains5′-biotin tag. The fragments attached to the beads are PCR amplifiedwithin droplets of an oil-water emulsion. The result is multiple copiesof clonally amplified DNA fragments on each bead. In the second step,the beads are captured in wells (pico-liter sized). Pyrosequencing isperformed on each DNA fragment in parallel. Addition of one or morenucleotides generates a light signal that is recorded by a CCD camera ina sequencing instrument. The signal strength is proportional to thenumber of nucleotides incorporated. Pyrosequencing makes use ofpyrophosphate (PPi) which is released upon nucleotide addition. PPi isconverted to ATP by ATP sulfurylase in the presence of adenosine 5′phosphosulfate. Luciferase uses ATP to convert luciferin tooxyluciferin, and this reaction generates light that is detected andanalyzed.

Another example of a DNA sequencing technique that can be used in themethods of the provided invention is SOLiD technology (AppliedBiosystems). In SOLiD sequencing, genomic DNA is sheared into fragments,and adaptors are attached to the 5′ and 3′ ends of the fragments togenerate a fragment library. Alternatively, internal adaptors can beintroduced by ligating adaptors to the 5′ and 3′ ends of the fragments,circularizing the fragments, digesting the circularized fragment togenerate an internal adaptor, and attaching adaptors to the 5′ and 3′ends of the resulting fragments to generate a mate-paired library. Next,clonal bead populations are prepared in microreactors containing beads,primers, template, and PCR components. Following PCR, the templates aredenatured and beads are enriched to separate the beads with extendedtemplates. Templates on the selected beads are subjected to a 3′modification that permits bonding to a glass slide. The sequence can bedetermined by sequential hybridization and ligation of partially randomoligonucleotides with a central determined base (or pair of bases) thatis identified by a specific fluorophore. After a color is recorded, theligated oligonucleotide is cleaved and removed and the process is thenrepeated.

Another example of a DNA sequencing technique that can be used in themethods of the provided invention is Ion Torrent sequencing (U.S. patentapplication numbers 2009/0026082, 2009/0127589, 2010/0035252,2010/0137143, 2010/0188073, 2010/0197507, 2010/0282617, 2010/0300559),2010/0300895, 2010/0301398, and 2010/0304982), the content of each ofwhich is incorporated by reference herein in its entirety. In IonTorrent sequencing, DNA is sheared into fragments of approximately300-800 base pairs, and the fragments are blunt ended. Oligonucleotideadaptors are then ligated to the ends of the fragments. The adaptorsserve as primers for amplification and sequencing of the fragments. Thefragments can be attached to a surface and is attached at a resolutionsuch that the fragments are individually resolvable. Addition of one ormore nucleotides releases a proton (H⁺), which signal detected andrecorded in a sequencing instrument. The signal strength is proportionalto the number of nucleotides incorporated.

Another example of a sequencing technology that can be used in themethods of the provided invention is Illumina sequencing. Illuminasequencing is based on the amplification of DNA on a solid surface usingfold-back PCR and anchored primers. Genomic DNA is fragmented, andadapters are added to the 5′ and 3′ ends of the fragments. DNA fragmentsthat are attached to the surface of flow cell channels are extended andbridge amplified. The fragments become double stranded, and the doublestranded molecules are denatured. Multiple cycles of the solid-phaseamplification followed by denaturation can create several millionclusters of approximately 1,000 copies of single-stranded DNA moleculesof the same template in each channel of the flow cell. Primers, DNApolymerase and four fluorophore-labeled, reversibly terminatingnucleotides are used to perform sequential sequencing. After nucleotideincorporation, a laser is used to excite the fluorophores, and an imageis captured and the identity of the first base is recorded. The 3′terminators and fluorophores from each incorporated base are removed andthe incorporation, detection and identification steps are repeated.Another example of a sequencing technology that can be used in themethods of the provided invention includes the single molecule,real-time (SMRT) technology of Pacific Biosciences. In SMRT, each of thefour DNA bases is attached to one of four different fluorescent dyes.These dyes are phospholinked. A single DNA polymerase is immobilizedwith a single molecule of template single stranded DNA at the bottom ofa zero-mode waveguide (ZMW). A ZMW is a confinement structure whichenables observation of incorporation of a single nucleotide by DNApolymerase against the background of fluorescent nucleotides thatrapidly diffuse in an out of the ZMW (in microseconds). It takes severalmilliseconds to incorporate a nucleotide into a growing strand. Duringthis time, the fluorescent label is excited and produces a fluorescentsignal, and the fluorescent tag is cleaved off. Detection of thecorresponding fluorescence of the dye indicates which base wasincorporated. The process is repeated.

Another example of a sequencing technique that can be used in themethods of the provided invention is nanopore sequencing (Soni G V andMeller A. (2007) Clin Chem 53: 1996-2001). A nanopore is a small hole,of the order of 1 nanometer in diameter. Immersion of a nanopore in aconducting fluid and application of a potential across it results in aslight electrical current due to conduction of ions through thenanopore. The amount of current which flows is sensitive to the size ofthe nanopore. As a DNA molecule passes through a nanopore, eachnucleotide on the DNA molecule obstructs the nanopore to a differentdegree. Thus, the change in the current passing through the nanopore asthe DNA molecule passes through the nanopore represents a reading of theDNA sequence.

Another example of a sequencing technique that can be used in themethods of the provided invention involves using a chemical-sensitivefield effect transistor (chemFET) array to sequence DNA (for example, asdescribed in US Patent Application Publication No. 20090026082). In oneexample of the technique, DNA molecules can be placed into reactionchambers, and the template molecules can be hybridized to a sequencingprimer bound to a polymerase. Incorporation of one or more triphosphatesinto a new nucleic acid strand at the 3′ end of the sequencing primercan be detected by a change in current by a chemFET. An array can havemultiple chemFET sensors. In another example, single nucleic acids canbe attached to beads, and the nucleic acids can be amplified on thebead, and the individual beads can be transferred to individual reactionchambers on a chemFET array, with each chamber having a chemFET sensor,and the nucleic acids can be sequenced.

Another example of a sequencing technique that can be used in themethods of the provided invention involves using a electron microscope(Moudrianakis E. N. and Beer M. Proc Natl Acad Sci USA. 1965 March;53:564-71). In one example of the technique, individual DNA moleculesare labeled using metallic labels that are distinguishable using anelectron microscope. These molecules are then stretched on a flatsurface and imaged using an electron microscope to measure sequences.

Additional detection methods can utilize binding to microarrays forsubsequent fluorescent or non-fluorescent detection, barcode massdetection using a mass spectrometric methods, detection of emittedradiowaves, detection of scattered light from aligned barcodes,fluorescence detection using quantitative PCR or digital PCR methods.

Single-Plex Assay Vs. Multi-Plex Assays

All of the previous and following embodiments can be performed as either‘single-plex’ or ‘multi-plex’ assays.

In one embodiment, a combined droplet is formed to allow “single-plex”binding to take place between target analyte and a first binding agentand a capture molecule. The single-plex binding interaction avoids anycross-reactivity which may be found when multiple binding agents aremixed together and may correspond to similar target analytes. FIG. 5depicts creation of single-plex assay. In FIG. 5. A) Two bindingreagents types are constructed: Barcoded Binders and Capture-TagBinders; B) Pairs of target-specific binders are made into a dropletlibrary (with ‘n’ elements), with each set of target binders in separatedroplets; C) The sample is made into sample droplets, and D) combinedwith the library droplets to initiate highly parallel ‘single-plex’binding reactions. After binding is complete, productive sandwiches areE) captured via the capture-tag (streptavidin (SA) biotin (B)interaction shown), and washed to remove unbound material; F) Thecaptured barcodes are released, recovered, and processed for reading; G)Reads for each barcode are counted (e.g. using sequencing). Anotherembodiment allows for multi-plex interaction within merged droplet. Inorder to create multi-plex assays, the first binding agent and secondbinding agents along with their target analyte have been tested andshown not to cross-react with a second, third, or N-number bindingagents and the corresponding second, third or N-number target analytesduring sandwich formation. In such embodiment, multiple binding agentsare within a droplet library and merged with a sample droplet, whereinmultiple target analyte sandwiches are formed with the same specificityas if the combining were performed as a single-plex assay.

In another exemplifying embodiment, a combined droplet is formed toallow “multi-plex” binding to take place between multiple targetanalytes and multiple first binding agents and multiple capturemolecules, i.e. multiple second binding agents with binding pairs. Asample droplet contains a first, second, . . . , N-number targetanalytes. A droplet library contains a first, second, . . . , N-numberfirst binding agents having an identifier that correspond to a first,second, . . . , N-number second binding agents. The droplets merge tocreate a first target analyte sandwich, a second target analytesandwich, . . . , a N-number target analyte sandwich. Afterimmobilization, the unbound sample is washed away, leaving multiplesandwich assays. The barcodes of all the assays are released andprocessed allowing for complex sample analysis.

In one embodiment, the methods previously described are used forquantification of individual proteins from homogenous liquids, includingbut not limited to bodily fluids, cell and tissue lystates, andbiochemical fractions. Within an embodiment, the amount of a targetprotein within a sample is determined using a binding agent with anidentifier and a capture molecule specific to different regions on atarget protein. The target protein specific identifier and capturemolecules represent a binding pair. The binding agent with theidentifier and the capture molecule attached to different epitopes onthe target protein, thus creating a sandwich complex. The identifiercontains indentifying information about specific protein, and in oneembodiment the identifier a barcode. The identifier may also containinformation about the paired capture molecule. Targeting two separateregions with binding pairs increases specificity of the sample. Thecapture molecule is immobilized to a solid support. Afterimmobilization, the sample is stringently washed removing unattachedsample, identifiers, and capture molecules. The remaining sandwichcomplexes highly correlate with the amount of targeted protein withinthe sample. An embodiment of the invention provides for releasing theidentifier from the sample, for example releasing a barcode by UVphotocleavage. The identifier is then counted using sequencing afterligation with NextGen sequencing adapters and sequencing primers. Othermethods of ligation may be applied to the identifier to prepare forsequencing. An example of a single target analyte sandwich is shown inFIG. 6A. The above method is not limited to single target proteins andallows for quantification of any single target analyte.

Another embodiment provides for quantification of protein complexes.Protein complexes includes any combination of two or more polypeptidechains, for example epidermal growth factor receptor dimers, a ribosome,a proteasome, a transcription activation pre-initiation complex.Utilizing the sandwich assay method previously described, the proteincomplex may be identified and counted using identifiers, in certainembodiments the identifiers include barcodes. In one embodiment, adroplet containing a targeted protein complex merged with a librarydroplet containing a capture molecule specific a first protein complexmember and a corresponding binding agent with an identifier specific tosecond first complex member suspected in the target protein complex. Asandwich is formed wherein the capture molecule is bound to the firstprotein complex member and the identifier is bound to a second proteincomplex member. The attached capture molecule then immobilizes thesandwich to a solid substrate, allowing for a stringent wash to removeall unbound sample, identifiers and capture molecules. The remainingsandwiches highly correlate to the amount of targeted protein complexeswithin the sample. The identifier is released. In one embodiment abarcode attached to the identifier is released using UV photocleavage.Once released, the barcode undergoes ligation with NextGen sequencingadapters and sequencing primers, or other suitable ligation techniques,and then counted using sequencing. An example of a complex sandwichassay is shown in FIG. 6B. The above method is not limited to proteincomplexes and allows for quantification of any complex analytecontaining more than one member.

In a further embodiment, complexes may also be identified and countedusing methods of the invention described above. Identifiers specific tocomplex members can contain identifying information about the targetcomplex member to which they attach (i.e. binder information), but alsocan contain identifying information relating to the capture molecule oradditional binders present in the library droplet. Therefore, the firstidentifier contains identifying information about the second complexmember and the capture molecule, and the second identifier containsindentifying information about a third protein complex member and thecapture molecule. An example of a complex analyte sandwich is shown inFIG. 6C (e.g. “3:1:2” in example C means Binder3 in the same droplet asBinder1 and Capture-Tag Binder2).

In a another embodiment, quantification of post-translationalmodifications from homogenous liquids including bodily fluids, cell andtissue lysates, and biochemical fractions. Post-translationalmodifications of proteins include phosphorylation, methylation,glycosylation, and ubiquitinylation and are critical components ofprotein function. Methods of the invention allow for identifying andquantifying specific post-translational modification proteins within asample. In one embodiment a binding agent is specific to a invariantepitope and the other biding agent is specific to a sequence-specificpost-translational modification. In one embodiment, a first bindingagent with an identifier, in one embodiment a barcode, is specific to aninvariant epitope on the target protein, and a capture molecule isspecific to sequence-specific post-translational modification. Whenintroduced to the target protein, the target protein forms a sandwichwith the first binding agent and the capture molecule. The sandwich isseparated from unattached samples and binding agents, wherein separationmay occur from immobilization followed by a stringent wash. Theremaining sandwiches highly correlate to the amount of target proteinshaving the post-translational modification. In one embodiment, theidentifier is released from the sandwich by a UV photocleavage and theidentifier is then used to analyze the target protein. In oneembodiment, the barcode is ligated using NextGen sequencing adapters andsequencing primers and then analyzes the target protein via sequencing.Example of a single sandwich wherein the target analyte is apost-translation modification is illustrated on FIG. 6D. Modificationsthat can be detected using the above methods are not limited topost-translational protein modifications, but apply to any modificationfor which a specific binding agent is available.

Methods of the invention also provide for quantification of individualnucleic acids from homogenous liquids (liquids including bodily fluids,cell and tissue lysate, and biochemical fractions). In one embodiment, aspecific region on a nucleic may be detected using methods of theinvention. A first binding agent is specific to a first region on thenucleic acid, and a second binding agent specific to the second regionon the nucleic acid. The first or the second binding agent may comprisea capture molecule or having an identifier. When the first and secondbinding agents attach to the corresponding regions on the target regionand a sandwich is produced. In one embodiment, a sandwich is created forSNP detection of DNA wherein the capture molecule is specific to atarget wild type sequence nearby the potential SNP-containing sequenceand the binding agent having a identifier is specific to potential SNP(see FIG. 6H). In another embodiment, a gene fusion is detected whereinthe capture molecule is specific to target sequences nearby thepotential fusion junction on gene 1 and the binding agent having aidentifier is specific to sequences found on gene 2 when fused to gene 1(see FIG. 6I). In another embodiment, full-length mRNA is detectedwherein the capture molecule is specific to the transcriptional startsequence region and the identifier is specific to the 3-prme end region(See FIG. 6F). In another embodiment, splice variants are detectedwherein a capture molecule is specific to the transcriptional startsequence region and the binding agent is specific to the splice variantregion (see FIG. 6G). In another embodiment, modified DNA (e.g.methylation or hydroxyl-methylation of cytosine) is detected using acapture molecule specific to a nearby DNA motif and a barcoded bindingagent specific for modified DNA seqeuences. Other embodiments of theinvention includes detecting untranslated RNA (including miRNA orlincRNA), and binding complexes of DNA to DNA, DNA to RNA, DNA or RNA toprotein (see FIG. 6E). In a further embodiment, introduction of acompetitive inhibitor increases specificity to ensure the binding agentor capture molecule are not binding on unspecific target regions(similar to the example in FIG. 7).

Embodiments of the invention further include using sandwich assays indroplets for quantification of proteins, nucleic acids, and othermolecules from single cells. An embodiment of the invention provides forencapsulating aseries of single cell containing droplets by usingcollections of single cells dispersed in liquid, for example a growthmedia or a phosphate buffered saline for droplet creation similar tomethods described above. In order to create droplets containing onlysingle cells, the concentration of single cells in a collection isdiluted to minimize multiple cell encapsulation. One embodiment of theinvention dilutes the single cell concentration to a level where onlyone cell is present for every 10 droplets formed. Once single celldroplets are formed, they are incubated in order to allow secretedmolecules of interest to accumulate inside the droplet, one can lyse thecell using reagents that release the cellular nucleic acids, proteins,and other components from cell compartments, or one can use immediatelyto analyze cell surface exposed material, or one can combine thesevarious analysis schemes. Each single cell droplet is combined with alibrary droplet in which a variety of assays may be conducted usingmethods of the invention including 1) analysis of secreted moleculesincluding but not limited to cytokines and growth factors by combiningviable single cells with a first binding agent and a second bindingagent specific to target secreted molecules; 2) analysis of cell surfacemolecules including but not limited to receptors and biomarkers bycombining a single cell with a droplet library containing a firstbinding agent and a second binding agent specific to target molecules;3) analysis of molecules released from single cells lysed insidedroplets including but not limited to cytoplasmic or nuclear proteins bycombining lysed single cell droplets with a first binding agent and asecond binding agent specific to a target intracellular molecule; 4) anycombination of the above assays.

Methods of the invention further provide for conducting further assayspreviously described for use with single cell droplets, includingquantification of individual proteins, protein post-translationalmodifications, protein complexes, protein/DNA complexes, and nucleicacids. In further embodiments, a combination of assays may be performed.In a non-limiting example, an assay may be performed by targeting bothsecreted molecules and cell surface molecules from the same cell. Inanother non-limiting example, a secreted target can be assayed beforecell lysis is induced, followed by cell lysis within the droplet andsubsequently assaying an intracellular target using additional bindingand capture agents in the library element, or by subsequent combinationwith a second library element droplet. Typical, but non-limiting methodsfor cell lysis within droplets include: 1) co-flowing a lysis buffer ina laminar flow alongside the incoming cell stream in the flow path justbefore the droplet-forming microfluidic nozzle; 2) introducing a lysisbuffer within the droplet library reagents; 3) use of a temperature orother inducible protease or lysis reagent; 4) mechanical abrasion insidedroplets traveling through microfluidic turns and constrictions; 5)laser-induced lysis. Library droplets used for single cell analysis cancontain paired binders for single-target analysis, or multiple bindingpairs for multi-plex target analysis (as long as the multiple bindingpairs retain sufficient specificity when in the same compartment). In apreferred embodiment, the single cell droplet contains a sampleidentifier and all individual molecule droplets dependent from thesample also have the same sample identifier. Therefore, the sampleidentifier combines with the target analyte identifier so all sandwichescan be traced to the sample.

In another embodiment, target analyte sandwiches are run on bulk sampleswithout the need for droplets, if cross reactivity or specificity ofbinding pairs in the presence of other binding pairs is not an issue. Insuch embodiment, a identifier library is created to analyze the targetanalytes within the sample without the use of droplets, and all relatedsteps needed for quantifying the analytes with use of barcodes also donot require droplets, i.e. capture occurs on beads in a bulk solutioninstead of in a droplet.

Libraries and Kits

proplet libraries are useful to perform large numbers of assays whileconsuming only limited amounts of reagents. A “droplet,” as used herein,is an isolated portion of a first fluid that completely surrounded by asecond fluid. In some cases, the droplets may be spherical orsubstantially spherical; however, in other cases, the droplets may benon-spherical, for example, the droplets may have the appearance of“blobs” or other irregular shapes, for instance, depending on theexternal environment. As used herein, a first entity is “surrounded” bya second entity if a closed loop can be drawn or idealized around thefirst entity through only the second entity.

In general, a droplet library is made up of a number of library elementsthat are pooled together in a single collection. Libraries may vary incomplexity from a single library element to 10¹⁵ library elements ormore. Each library element is one or more given components at a fixedconcentration. Each droplet includes a first binding agent having adifferentially detectable identifier and a second binding agent. Thebinding agents may be any of the agents described above. Each dropletmay further include a sample identifier that can bind to the identifierlinked to the first binding agent. In this manner, each droplet includesan identifier for a particular target analyte and an identifier for aspecific droplet. Each droplet may further include a competitiveinhibitor. The terms “droplet library” or “droplet libraries” are alsoreferred to herein as an “emulsion library” or “emulsion libraries.”These terms are used interchangeably throughout the specification.

The droplets range in size from roughly 0.5 micron to 500 micron indiameter, which corresponds to about 1 pico liter to 1 nano liter.However, droplets can be as small as 5 microns and as large as 500microns, Preferably, the droplets are at less than 100 microns, about 1micron to about 100 microns in diameter. The most preferred size isabout 20 to 40 microns in diameter (10 to 100 picoliters). The preferredproperties examined of droplet libraries include osmotic pressurebalance, uniform size, and size ranges.

The droplets comprised within the droplet library provided by theinstant invention may be uniform in size. That is, the diameter of anydroplet within the library will vary less than 5%, 4%, 3%, 2%, 1% or0.5% when compared to the diameter of other droplets within the samelibrary. The uniform size of the droplets in the library is critical tomaintain the stability and integrity of the droplets and is alsoessential for the subsequent use of the droplets within the library forthe various biological and chemical assays described herein.

The droplet libraries of the present invention are very stable and arecapable of long-term storage. The droplet libraries are determined to bestable if the droplets comprised within the libraries maintain theirstructural integrity, that is the droplets do not rupture and elementsdo not diffuse from the droplets. The droplets libraries are alsodetermined to be stable if the droplets comprised within the librariesdo not coalesce spontaneously (without additional energy input, such aselectrical fields described in detail herein). Stability can be measuredat any temperature. For example, the droplets are very stable and arecapable of long-term storage at any temperature; for example, e.g., −70°C., 0° C., 4° C., 37° C., room temperature, 75° C. and 95° C.Specifically, the droplet libraries of the present invention are stablefor at least 30 days. More preferably, the droplets are stable for atleast 60 days. Most preferably, the droplets are stable for at least 90days.

In certain embodiments, the present invention provides an emulsionlibrary comprising a plurality of aqueous droplets within an immisciblefluorocarbon oil comprising at least one fluorosurfactant, wherein eachdroplet is uniform in size and comprises the same aqueous fluid andcomprises a different library element. The present invention alsoprovides a method for forming the emulsion library comprising providinga single aqueous fluid comprising different library elements,encapsulating each library element into an aqueous droplet within animmiscible fluorocarbon oil comprising at least one fluorosurfactant,wherein each droplet is uniform in size and comprises the same aqueousfluid and comprises a different library element, and pooling the aqueousdroplets within an immiscible fluorocarbon oil comprising at least onefluorosurfactant, thereby forming an emulsion library.

For example, in one type of emulsion library, the first and secondbinding agents are pooled in a single source contained in the samemedium. After the initial pooling, the first and second binding agentsare then encapsulated in droplets to generate a library of dropletswherein each droplet includes a different set of first and secondbinding agents. The dilution of the initial solution enables theencapsulation process. In some embodiments, the droplets formed willeither contain a single set of first and second binding agents or havingnothing, i.e., be empty. In other embodiments, the droplets formed willcontain multiple sets of first and second binding agents so thatmultiplexing may be performed in each droplet.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

1. A method for identifying a target analyte, the method comprising:forming one or more compartmentalized portions of fluid, each comprisinga target analyte, a first binding agent comprising a target identifier,and a second binding agent specific to the target analyte underconditions that produce a complex of the first and second binding agentswith the target analyte; separating the complexes; and detecting thetarget identifier, thereby detecting the target analyte.
 2. The methodaccording to claim 1, further comprising the step of counting the targetidentifier.
 3. The method according to claim 1, wherein each of the oneor more compartmentalized portions comprises a different targetidentifier.
 4. The method according to claim 1, wherein thecompartmentalized portion is a droplet.
 5. The method according to claim4, wherein the droplet is in a microchannel.
 6. The method according toclaim 4, wherein the droplet is surrounded by an immiscible carrierfluid.
 7. The method according to claim 6, wherein the carrier fluid isoil.
 8. The method according to claim 1, wherein the forming stepcomprises merging a droplet comprising either the first and/or thesecond binding agents with a droplet comprising the target analyte. 9.The method according to claim 1, wherein the forming step comprisesflowing a droplet comprising either the first and/or the second bindingagents such that it interacts with a fluid stream comprising the targetanalyte to cause a portion of the fluid stream to integrate with thedroplet.
 10. The method according to claim 1, wherein thecompartmentalized portion further comprises a competitive bindinginhibitor.
 11. The method according to claim 1, wherein the targetidentifier is releasably attached to the first binding agent.
 12. Themethod according to claim 1, wherein a portion of the second bindingagent is functionalized for immobilization to a support.
 13. The methodof claim 12, wherein the support is a solid support.
 14. The method ofclaim 12, wherein the portion of the second binding agent is a terminalportion of the second binding agent.
 15. The method according to claim1, wherein the separating step comprises: immobilizing the complexes toa support via the functionalized portion of the second binding agent;and releasing contents from the compartmentalized portion.
 16. Themethod of claim 11, further comprising the step of separating targetidentifiers not in a complex from target identifiers in a complex. 17.The method according to claim 1, wherein the separating step comprises:releasing the contents from the compartmentalized portion; immobilizingthe complexes to the solid support via the functionalized portion of thesecond binding agent; and washing the released contents to removeunbound sample components.
 18. The method according to claim 1, whereinthe target analyte is derived from a sample and wherein thecompartmentalized portion further comprises an identifierof the sample.19. The method of claim 18, wherein the identifier of the sample iscapable of complexing with the target identifier.
 20. The methodaccording to claim 1, wherein the target identifier is a barcodeoligonucleotide.
 21. The method according to claim 20, wherein thebarcode oligonucleotides are released from the first binding agentsafter the complexes have been separated.
 22. The method of claim 21,wherein the barcode oligonucleotides are amplified.
 23. The methodaccording to claim 21, wherein a plurality of the released targetidentifiers are attached to each other to form a nucleic acid strand.24. The method according to claim 22 or 23, wherein the strand isdetected.
 25. The method of claim 24, wherein the strand is sequencedfor detection.
 26. The method according to claim 25, wherein sequencingis sequencing-by-synthesis.
 27. The method according to claim 26,wherein sequencing-by-synthesis is single moleculesequencing-by-synthesis.
 28. An emulsion library comprising a pluralityof droplets, wherein each droplet comprises, in an aqueous phase fluid,a first binding agent with a target identifier and a second bindingagent.
 29. The library according to claim 28 wherein the first andsecond binding agent both bind the same target analyte.
 30. The libraryaccording to claim 29, wherein a portion of the second binding agent isfunctionalized for immobilization to a solid support.
 31. The libraryaccording to claim 28, further comprising a bead.
 32. The libraryaccording to claim 28, further comprising a competitive inhibitor. 33.The library according to claim 28, further comprising a sampleidentifier.
 34. The library according to claim 28, wherein the pluralityof droplets are within an immiscible fluid.
 35. The library of claim 34,wherein the immiscible fluid is an oil.
 36. The library according toclaim 34, wherein the oil is a fluorinated oil.
 37. The libraryaccording to claim 34, wherein the oil comprises a surfactant.
 38. Thelibrary according to claim 37, wherein the surfactant is afluorosurfactant.
 39. The library according to claim 28, wherein thetarget identifier is a barcode oligonucleotide.
 40. The libraryaccording to claim 28, wherein the first and second binding agent areindependently selected from the group consisting of a nucleic acid, aprotein, an antibody, an aptamer, a small molecule, a lipid, a sugar, orcombinations thereof.
 41. The library according to claim 28, whereineach droplet comprises multiple different sets of first and secondbinding agents.
 42. A method for detecting a target nucleic acid, themethod comprising: forming a compartmentalized portion of fluidcomprising a sample containing a target nucleic acid, a first bindingagent comprising a target identifier, and a second binding agentspecific to the target nucleic acid under conditions that produce acomplex of the first and second binding agents with the target nucleicacid; separating the complexes; and detecting the complex, therebydetecting the target nucleic acid.
 43. The method of claim 42, whereinthe target nucleic acid is a splice variant and the first binding agentand second binding agent are selective for detection of the splicevariant.
 44. The method of claim 42, wherein the target nucleic acid isa fusion product comprising a first and second gene product joined at afusion junction, the first binding agent is specific to the targetsequence on the second gene nearby the suspected fusion junction, andthe second binding agent is specific to a target sequence on a firstgene nearby the fusion junction.
 45. The method of claim 42, wherein thetarget nucleic acid comprises a single nucleotide polymorphism, thefirst binding agent is specific to the single nucleotide polymorphism,and the second binding agent is specific to a portion of wild-typesequences in proximity to the single nucleotide polymorphism.
 46. Themethod according to claim 42, wherein the target identifier is a barcodeoligonucleotide.
 47. The method according to claim 46, wherein thebarcode oligonucleotides are released from the first binding agentsafter the complexes have been separated from the unbound samplecomponents.
 48. The method according to claim 47, wherein a plurality ofthe released target identifiers are attached to each other to form atemplate nucleic acid strand.
 49. The method according to claim 48,wherein the strand is amplified.
 50. The method according to claim 48,wherein the strand is sequenced.
 51. The method according to claim 50,wherein sequencing is sequencing-by-synthesis.
 52. The method accordingto claim 51, wherein sequencing-by-synthesis is single moleculesequencing-by-synthesis.
 53. A method for detecting a target analyte,the method comprising: forming a compartmentalized portion of fluidcomprising a portion of a sample suspected of containing a targetanalyte and unspecific analytes, a first binding agent comprising atarget identifier, a second binding agent specific to the targetanalyte, and a competitive inhibitor specific to unspecific analytesunder conditions that produce a complex of the first and second bindingagents with the target analyte; separating the complexes; and detectingthe complex, thereby detecting the target analyte.
 54. The method ofclaim 53, wherein the compartmentalized portion is a droplet.
 55. Themethod according to claim 54, wherein the droplet is present in amicrochannel.
 56. The method according to claim 54, wherein the dropletis surrounded by an immiscible carrier fluid.
 57. The method accordingto claim 56, wherein the carrier fluid is oil.
 58. The method accordingto claim 45, wherein the compartmentalized portion further comprises asample identifier that binds the target identifier.
 59. The methodaccording to claim 58, wherein the target identifier is a barcodeoligonucleotide.
 60. The method according to claim 59, wherein thebarcode oligonucleotides are released from the first binding agentsafter the complexes have been separated from the unbound samplecomponents.
 61. The method according to claim 60, wherein a plurality ofthe released target identifiers are attached to each other to form atemplate nucleic acid strand.
 62. The method according to claim 61,wherein the strand is amplified.
 63. The method according to claim 62,wherein the strand is sequenced.
 64. A method for identifying a targetanalyze, the method comprising: providing a target analyte, a firstbinding agent comprising a target identifier, and a second binding agentspecific to the target analyte under conditions that produce a complexof the first and second binding agents with the target analyte;separating the complexes; and detecting the target identifier, therebydetecting the target analyte.
 65. The method of claim 64, wherein thedetecting step comprises sequencing a nucleic acid, hybridizing to aprobe, reading an electromagnetic signal, reading emitted waves orparticles, and/or detecting by use of mass signature.